Single domain antibodies for inhibiting neutrophil elastase activity

Abstract

The present invention relates to a binding agent capable of specifically binding and competitively inhibiting neutrophil elastase, the binding agent comprising an immunoglobulin single variable domain (ISVD), as well as related products, methods and uses, for example, methods and uses, particularly for the prevention, treatment or diagnosis of inflammatory diseases.

Description

Technical Field

[0001] This invention is broadly applicable to the medical field, and particularly relates to reagents, more specifically, binders and pharmaceutical preparations suitable for inhibiting neutrophil elastase (NE) activity or detecting / quantifying NE activity. Background Technology

[0002] Neutrophil elastase (NE) is a protease that plays a crucial role in immune responses and host defense mechanisms under physiological and disease-related conditions. NE is stored in azurophilic granules of polymorphonuclear nucleophiles and can be released during neutrophil degranulation and neutrophil extracellular trap release (NETosis). Under physiological conditions, the amount of extracellular NE is tightly regulated, primarily by antiproteases AAT (α1-antitrypsin) and Elafin. However, under many pathological conditions, this balance is disrupted, leading to excessively high levels of extracellular NE, which is associated with progressive tissue damage, loss of related functions, and impaired immune system regulation. Various types of inhibitors, including protein-based inhibitors and small molecule inhibitors, are known to inhibit NE activity. Regarding protein-based inhibitors, inhaled AAT (an endogenous glycoprotein inhibitor of neutrophil elastase) has been tested for the treatment of respiratory pathologies. However, the effectiveness of inhaled AAT in reducing inflammatory parameters and improving lung function is very limited. While it does not reduce the frequency of infection exacerbations, it can alleviate their severity. The need for high-dose inhaled AAT reflects the protein’s inadequacy (Amin and Ratjen, Expert Opin Emerg Drugs 2014, vol. 19(1), 143-55). This inadequacy may be attributed to the rapid degradation of AAT in lung tissue, as this glycoprotein is highly unstable in biological fluids due to its susceptibility to proteolysis and oxidation (Suter et al., Eur Respir J. 1991, vol. 4(1), 40-9). Furthermore, the use of nebulizers for pulmonary administration to accommodate high doses of AAT requires prolonged operation and inhalation periods, further increasing the treatment burden on patients. Other disadvantages associated with AAT include increased cost and limited production capacity, as it is derived from human plasma through a purification process. In addition, small-molecule inhibitors of NE have been extensively studied. However, due to significant organ toxicity, only a few of these molecules (e.g., Sivelestat) have been approved for marketing as drugs for a limited number of conditions. The challenges in developing small molecule drugs include poor efficacy in inhibiting elastase, lack of specificity for elastase, and insufficient metabolic stability in vivo. Therefore, there is an urgent need to develop novel, potent drugs that effectively inhibit norepinephrine (NE) for patients whose clinical needs are not yet fully met. Summary of the Invention

[0003] This invention is based, at least in part, on the identification of an immunoglobulin single variable domain (ISVD) binder that binds to and effectively inhibits neutrophil elastase (NE). Specifically, as shown in the experimental section of this specification, the NbE201 ISVD demonstrated in the embodiments of this invention is a competitive inhibitor of NE, exhibiting high affinity, high specificity, and high inhibitory capacity for NE. Furthermore, the ISVD exhibits high stability and a long half-life in the lungs, especially after PEGylation. This paves the way for diagnostic, prognostic, and therapeutic applications using the ISVD disclosed herein.

[0004] In view of these advantages, one aspect of the present invention provides a binder capable of specifically binding to and inhibiting neutrophil elastase (NE), wherein the binder comprises an immunoglobulin single variable domain (ISVD), and the binder competes with elastase inhibitor 3 (EI3) for binding to NE.

[0005] Another aspect of the present invention provides a binding agent capable of specifically binding to and inhibiting neutrophil elastase (NE), wherein the binding agent comprises an immunoglobulin single variable domain (ISVD) that binds to at least one of the R36, A60, N61, P96, V97, S195, G218, and G219 residues of NE, preferably binding to two or more of the R36, A60, N61, P96, V97, S195, G218, and G219 residues, more preferably binding to three or more of the R36, A60, N61, P96, V97, S195, G218, and G219 residues of NE, and even more preferably binding to all of the R36, A60, N61, P96, V97, S195, G218, and G219 residues of NE, wherein these residues are annotated according to the chymotrypsinogen protocol. In some implementations, ISVD binds to at least residues R36, A60, N61, P96, V97, and S195 of NE.

[0006] Another aspect of the invention provides a binding agent capable of specifically binding to and inhibiting neutrophil elastase (NE), wherein the binding agent comprises an ISVD having a complementarity-determining region 1 (CDR1) having the sequence shown in SEQ ID NO: 1 (GRTISLYR), a CDR2 having the sequence shown in SEQ ID NO: 2 (INWSGDMT), and a CDR3 having the sequence shown in SEQ ID NO: 3 (TADPKLLPLADSSYGY).

[0007] On the other hand, a binding agent capable of specifically binding to and inhibiting NE is provided, wherein the binding agent comprises an ISVD, the ISVD comprising CDR1, CDR2 and CDR3, each present in SEQ ID NO: 4, wherein CDR1, CDR2 and CDR3 are annotated according to any one of the numbering systems IMGT, Kabat, Chlotia, Martin or AHo.

[0008] One relevant party provides a nucleic acid molecule containing a polynucleotide sequence encoding the aforementioned binding agent, or provides a vector containing the nucleic acid molecule.

[0009] One related aspect provides a cell or virus comprising a nucleic acid molecule or vector, optionally wherein the cell is capable of expressing or expressing a binding agent, or the virus is configured to induce recipient cells infected by the virus to express the binding agent.

[0010] One related aspect provides a pharmaceutical composition comprising a binder or nucleic acid molecule or carrier or cell or virus, and a pharmaceutically acceptable carrier and / or diluent and / or excipient, optionally, the pharmaceutical composition comprising other inhibitors of NE.

[0011] An additional aspect provides kits, such as diagnostic kits, containing a binder or nucleic acid molecule or vector or cell or virus. Other aspects provide a method for determining the level of active neutrophil elastase in a sample, the method comprising contacting the sample with a binder and detecting at least the neutrophil elastase bound to the binder.

[0012] On the other hand, it provides the binders, nucleic acid molecules, carriers, cells, viruses or pharmaceutical compositions as described above for pharmaceutical use.

[0013] On the other hand, it provides the binders, nucleic acid molecules, carriers, cells, viruses or pharmaceutical compositions as described above for use in the prevention or treatment of inflammatory diseases.

[0014] One related aspect provides a method for treating an inflammatory disease in a subject requiring treatment, comprising administering to the subject an effective amount of a binder, nucleic acid molecule, carrier, cell, virus, or pharmaceutical composition as described above.

[0015] The binder of the present invention can also be used to detect active NE. Other methods of the present invention also provide a method for determining the level of active neutrophil elastase in a sample, the method comprising contacting the sample with the above-described binder and detecting at least the neutrophil elastase bound to the binder.

[0016] On the other hand, a method for diagnosing, prognosing, and / or monitoring inflammatory diseases in subjects is provided, comprising contacting a biological sample obtained from a subject with a binding agent as described above, and determining the level and / or activity of neutrophil elastase in the sample by detecting at least the neutrophil elastase bound to the binding agent.

[0017] These and other aspects and preferred embodiments of the invention will be described in the following sections and the appended claims. The subject matter of the appended claims is hereby expressly incorporated into this specification. Attached Figure Description

[0018] The following description of the accompanying drawings of specific embodiments of the present invention is merely exemplary in nature and is not intended to limit the content, application, or use of the present invention.

[0019] Figure 1 The sequence of mature hNE in the FASTA scheme is matched with the numbering scheme for chymotrypsinogen. Residues of the catalytic triad (H57, D102, and S195) are shown with white letters on a dark gray background. An asterisk indicates a glycosylation site (N...). 109 and N 204). Residues marked with light gray letters and background correspond to gaps (R147-N148) in the crystal structure due to low resolution. The amino acids involved in binding with Nb E201 are R36, A60, N61, P96, V97, S195, G218, and G219, marked with hash marks. Binding with S195 is achieved via water molecules.

[0020] Figure 2 The sequence of .ISVD NbE201 (SEQ ID NO: 4) shows the CDR1, CDR2 and CDR3 sequences annotated according to the IMGT, Kabat, Chlotia, Martin or AHo numbering system.

[0021] Figure 3A) Quantification of the surface percentage of alveolar-capillary lesions after intranasal instillation of hNE. All lung tissue sections stained with hematoxylin / eosin were digitized and images were analyzed using Visiopharm® software. The following four steps were performed: 1) outlining the lung tissue (black); 2) excluding the bronchi and capillary analysis areas; 3) outlining the alveolar tissue (gray) and alveoli (white); and 4) outlining the lesion areas (arrows). Protective effect of nanobodies against HNE-induced acute lung injury syndrome in a mouse model. Acute lung injury syndrome was identified by the following: B) presence of hemoglobin in bronchoalveolar lavage fluid, C and D) presence of erythrocytes on the alveolar surface (black arrows). Data are presented as mean ± standard deviation (SD), n = 3–12 mice / group. AAT: α-antitrypsin; HNE: human neutrophil elastase; Nb: nanobody

[0022] Figure 4 Residual hNE activity (expressed as a percentage) after hydrolysis of the small chromogenic substrate N-succinyl-Ala-Ala-Ala-pNA (1 mM) at different [inhibitor] / [hNE] ratios. The inhibitors tested are as follows: The inhibitory effect of NbE201 on hNE (N=3), The inhibitory effect of AAT on hNE (N=1), The inhibitory effect of EI3 (elastase inhibitor 3 or MeOSuc-AAPV-CMK) on hNE (N=2) and ( The inhibitory effect of cefotaxime on hNE (N=2). The concentration of hNE was 50 nM. The points shown are the average of independent measurements, and the error bars represent the standard deviation (SD). The data were fitted to a single exponential equation using SigmaPlot software for visualization.

[0023] Figure 5 Residual hNE activity (expressed as a percentage) after elastin hydrolysis at different [inhibitor] / [hNE] ratios. The inhibitory effect of AAT on hNE (N=3), The inhibitory effect of EI3 on ​​hNE (N=2), The inhibitory effect of NbE201 on hNE (N=3) and ( The inhibitory effect of seviloside on hNE (N=2). The points shown are the average of independent measurements, and the error bars correspond to the standard deviation.

[0024] Figure 6Representative measurements of the affinity between NbE201 and hNE were performed using BLI. On the x-axis, time is expressed in seconds. On the y-axis, the interference signal is expressed in nm. The vertical dashed line separates the binding and dissociation steps. The binding and dissociation kinetics between different concentrations of hNE (analyte) and NbE201 (ligand) immobilized on the sensor are shown. Each curve (black) represents the measurement data for a single concentration of hNE used. The values ​​are: 25 nM, 6.25 nM, 3.125 nM, 1.563 nM, and 0.753 nM (from top to bottom). The gray area represents the fitted data from which K is derived. D Values ​​and kinetic parameters. Data were fitted to a 1:1 ligand model using Octet software version 10.0 (Satorius). Two independent experimental replicates (N=2) were performed, and the mean (± standard deviation) of the kinetic parameters were measured. The results are shown in Table 2.

[0025] Figure 7 The residual activities of several serine proteases in the presence of different concentrations of NbE201. A) The protease tested was human protease 3 (hPR3, Human cathepsin G (hCG) ) and porcine pancreatic elastase (pPE, B) Mouse elastase (mNE, The experiment was repeated three times (N=3). The points shown are the average of independent measurements, and the error bars represent the SD.

[0026] Figure 8 Sensing diagrams of the binding and dissociation steps of NbE201 interactions with different serine proteases. 0 to 70 seconds: binding step; over 70 seconds: dissociation step. hCG( The sequence identity with hNE was 36.7%, hPR3 ( The sequence identity with hNE was 54.7%), mPE (□, sequence identity was 36.8%), and pPE (○, sequence identity with hNE was 39.2%).

[0027] Figure 9Representative sensor plots of the binding and dissociation steps of the interaction between NbE201 and different concentrations of mNE. On the x-axis, time is expressed in seconds. On the y-axis, the interference signal is expressed in nm. Dashed lines separate the binding and dissociation steps. The black portion represents the experimental signal. The gray portion represents the fitting using a 1:1 model. From top to bottom, the tested mNE concentrations were 100 – 50 – 25 – 12.5 – 6.25 – 3.125 nM. Binding and dissociation kinetics were measured for 300 seconds and 600 seconds, respectively. The obtained sensor plots were fitted to a 1:1 model using Octet software version 10.0 (Sartorius) to derive the binding rate constant (k). on ) and dissociation rate constant (k off and equilibrium dissociation constant (K) D =k off / k on An experiment was conducted, and the measured kinetic parameters are shown in Table 2.

[0028] Figure 10 Competitive binding between NbE201 and hNE complexed with a series of known inhibitors was monitored using a BLI sensor. Top left panel: Free hNE can bind to NbE201 immobilized on the BLI sensor; Bottom left panel: hNE complexed with competitive inhibitors cannot bind to NbE201 immobilized on the BLI sensor. The competitive inhibitors tested were: (i) Nb-H7S-Nter-P, hNE inhibitor III (MeOSuc-Ala-Ala-Pro-Val-CMK), and (ii) AAT. A control experiment was conducted using only hNE. After hydration, biotinylated ISVD NbE201 (10 µg·mL⁻¹) was first added. -1 The sensor was immobilized on a streptavidin (SA) sensor. In the binding step (left figure), the sensor was immersed in an hNE (250 nM) solution or an hNE solution combined with AAT, Nb-H7S-Nter-P1, or hNE inhibitor III (I). The right figure records the results using only hNE (SA). ) and in AAT ( hNE inhibitor III ) or Nb-H7S-Nter-P1( The sensor image under the presence of )

[0029] Figure 11 Monitoring urea-induced NbE₂O₁ evolution and conversion using intrinsic fluorescence and far-ultraviolet circular dichroism (far-UV-CD). A) Monitoring the intensity change at 337 nm. B) Monitoring the evolution and conversion using the maximum intensity wavelength (λ). maxThe conversion of changes in ) is monitored. The solid line represents the best fit calculated using Equation 1. C) Natural intrinsic fluorescence spectrum ( ) and hNE denaturation ( (D) Status. The conversion is monitored via far-UV-CD.

[0030] Figure 12 The urea-induced NbE201 expansion transformation was normalized using Equation 2. Relative to the expanded gradation curve of the CD signal, ( The expanded series curve relative to the λmax signal, and ( The expanded fractional curve relative to the 337 nm signal.

[0031] Figure 13 An example of monitoring the denaturation and regeneration curves of NbE2O1 at 206 nm using far-UV-CD. The denaturation transition curve from 25 to 97°C, The annulation transition curve from 97°C to 25°C is shown. The inset shows the spectrum of NbE₂O₁ in the far UV CD (190–250 nm). The spectrum of NbE2O1 at 25°C, The spectrum of NbE201 at 90°C and ( The spectrum of NbE201 after denaturation / renaturation at 25°C.

[0032] Figure 14 The thermally induced unfolding transformation of NbE2O1 was monitored by changes in far-UV-CD at 206 nm and normalized using Equation 2. Each symbol represents a transformation for a different production batch: ( Production batch 18032021, ( Production batch 07052021 and ( Production batch 26022021, with a total of N=3 duplicates.

[0033] Figure 15 Stability of the NbE201+hNE complex over time. The integrity of NbE201 was monitored over a 14-day period and analyzed by SDS-PAGE (top curve). The bottom curve shows the residual activity of hNE monitored over 14 days.

[0034] Figure 16The three-dimensional structure of the NbE201+hNE complex was resolved using X-ray crystallography. A) shows the NbE201 / hNE complex, revealing the contact between amino acids P100, K101, D107, Y110, and Y112 (black letters) of CDR3 in NbE201 and hNE (A60, R36, N61, and P96 in light gray letters). B) shows the polar interaction between NbE201 (black S30) and hNE (light gray G218 and G219). The hydrophobic interaction between V2 of NbE201 and V97 of hNE is shown in black. These figures were created using PyMol software. The labeling of hNE residues followed the chymotrypsinogen protocol (Hartley, BS (1970), doi: 10.1098 / rstb.1970.0010). C) shows the structure of NbE201 alone in bands. These images were created using PyMol software.

[0035] Figure 17 The three-dimensional structure of the NbE201+hNE complex was resolved using X-ray diffraction. A) shows the interaction sites between hNE and NbE201 CDRs. CDR1, CDR2, and CDR3 are shown in dark gray. The catalytic tertiary (H57, D102, S195) is shown in dark gray. B) shows the surface of hNE, revealing that NbE201 CDR3 penetrates the crack containing the active site of hNE. Residue S195 in the catalytic tertiary contacts residue P100 in NbE201 CDR3 via water molecules (indicated by arrows).

[0036] Figure 18 Inhibitory effect of PEGylated and non-PEGylated NbE201 on hNE. Suc-Ala-Ala-Pro-Val-pNA was degraded with purified hNE (0.169 µM) within one hour with or without PEGylated (linear PEG10 and PEG20 or branched PEG40) or non-PEGylated NbE201, followed by absorbance measurements. hNE:Nb molar ratio = 1:0.25, 1:0.5, 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5. N=3, n=3. Mean ± SD.

[0037] ( NbE201, NbE201-PEG10 ), NbE201-PEG20 ( ) and NbE201-PEG40 ( ).

[0038] Figure 19Residual activity of hNE was measured before or after ISVD exposure to atomization, magnetic stirring for 1 or 4 hours, or 5 or 10 freeze-thaw (F / T) cycles in the presence of NbE201 (-PEG10 or 40) (control). Suc-Ala-Ala-Pro-Val-pNA was degraded with purified hNE within one hour in the presence of PEGylated (PEG10 or 40) or non-PEGylated NbE201, followed by absorbance measurements. hNE:Nb molar ratio = 1:1. N=3, n=3 (NbE201 wt and NbE201-PEG40). N=2, n=3 (NbE201-PEG10). Mean ± SD. Statistical tests: One-way ANOVA followed by Dunnett's post-hoc test, compared with the control group. P-value < 0.05 p-value < 0.01 p-value < 0.001. The experiment was performed twice using NbE201-PEG10, three times using NbE201 and NbE201-PEG40, and was technically replicated three times.

[0039] Figure 20 Inhibitory effect of NbE201 on sputum protein hydrolysis activity: inhibition curve. Within one hour in the presence or absence of non-PEGylated NbE201, via 20 mg of purulent sputum from cystic fibrosis patients (… ) and mucopurulent sputum ( Suc-Ala-Ala-Pro-Val-pNA was degraded, and then absorbance was measured. N=3, n=3. A representative experiment from three series of experiments. Mean ± SD of three technical replicates.

[0040] Figure 21 Inhibitory effect of NbE201-(PEG10 or 40) or AAT on sputum protein hydrolysis activity: inhibition curves. In the presence or absence of increasing concentrations of non-PEGylated NbE201 (PEG10 or 40) ), NbE201-PEG10 ( ), NbE201-PEG40 ( ) or AAT( Within one hour, Suc-Ala-Ala-Pro-Val-pNA was degraded using 15 mg of sputum from cystic fibrosis patients, followed by absorbance measurements. N=3, n=3. One representative experiment from three series. Mean ± SD of three technical replicates. Statistical tests: one-way ANOVA followed by Tukey post-hoc test.

[0041] Figure 22The molar ratio of nanobody / AAT:hNE was used to inhibit 50% of sputum proteolytic activity. Nonlinear regression curves were fitted using GraphPadPrism 9.1.2 to interpolate Nb / AAT concentrations, thereby inhibiting 50% of the activity in 10–15 mg cystic fibrosis sputum. The molar ratio of Nb / AAT:hNE in sputum was assessed by evaluating elastase activity in different sputum samples using a standard curve of purified hNE. N=4, n=3, where N represents the number of experiments and n represents the number of replicates. Different symbols for each data point in the graphs correspond to different sputum samples (one symbol corresponds to one sputum sample). Statistical tests: One-way ANOVA was performed on paired data, followed by Dunnett's post-hoc test, compared with NbE201. P-value < 0.05

[0042] Figure 23 Inhibition of sputum proteolytic activity by PEGylated and non-PEGylated NbE201 (0 to 24 hours). Suc-Ala-Ala-Pro-Val-pNA was degraded via 20 mg sputum from cystic fibrosis patients within one hour of exposure to 7.5 µg (0.52 nmol) of PEGylated (linear PEG10 or branched PEG40) or non-PEGylated NbE201 at 0, 1, 4, or 24 hours, followed by absorbance measurements. Control conditions: no restriction on elastase activity in sputum. N=3, n=3. Mean ± SD of 3 technical replicates. One representative experiment. N represents the number of experiments; n represents the number of replicates. (Comparison) NbE201-PEG10, ( )NbE201, and ( )NbE201-PEG40.

[0043] Figure 24 Inhibition of sputum proteolytic activity by AAT, PEGylated and non-PEGylated NbE201 (0 to 24 hours). Within one hour of exposure to 0.56 nmol of PEGylated (linear PEG10 or branched PEG40), wild-type NbE201, or AAT, 15 mg of sputum from cystic fibrosis (CF) patients was used to degrade Suc-Ala-Ala-Pro-Val-pNA, followed by absorbance measurements. N=2, n=3. Mean ± SD of 3 technical replicates. One representative experiment. (Comparison) NbE201-PEG10, ( NbE201, ( NbE201-PEG40, and ( AAT. N represents the number of experiments; n represents the number of repetitions.

[0044] Figure 25 Inhibitory effect of NbE201(-PEG40) or AAT on the proteolytic activity of sputum in COPD (Chronic Obstructive Pulmonary Disease): Inhibition curves. Suc-Ala-Ala-Pro-Val-pNA was degraded by 15 mg sputum from COPD patients within one hour in the presence or absence of ascending concentrations of wild-type NbE201, NbE201-PEG40, or AAT, followed by absorbance measurements. N=1, n=2. Mean ± SD of two technical replicates. NbE201, ( NbE201-PEG40, ( AAT.N represents the number of experiments; n represents the number of repetitions.

[0045] Figure 26 Inhibitory effect of NbE201(-PEG40) or AAT on the proteolytic activity of sputum with ciliary dyskinesia: inhibition curves. Suc-Ala-Ala-Pro-Val-pNA was degraded by 8 mg of sputum within one hour in the presence or absence of increasing concentrations of NbE201, NbE201-PEG40, or AAT, followed by absorbance measurements. N=1, n=3. Mean ± SD of 3 technical replicates. NbE201, ( NbE201-PEG40, ( )AAT.

[0046] Figure 27 Percentage of initial doses of NbE201, NbE201-PEG40, or AAT harvested at 0 hours following intratracheal instillation in Swiss or β-ENaC mice. N=5. Mean ± SD. Statistical tests: one-way ANOVA, post-hoc tests: Tukey. p-value < 0.01 p-value < 0.001 p-value < 0.0001. NbE201 Swiss mice, NbE201-PEG40 Swiss mice NbE201 β-ENaC mice, NbE201-PEG40 β-ENaC mice, AAT Swiss mice, and AAT β-ENaC mice. Detailed Implementation

[0047] As used herein, the singular forms a (a), an (an), and the (the) include both singular and plural references, unless the context clearly indicates otherwise.

[0048] As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “having,” “have,” “including,” “includes,” or “containing,” and are inclusive or open-ended, not excluding other members, elements, or method steps not listed. These terms also include “constituting,” “essentially comprising,” and “comprises,” as well as terms such as “mainly comprising,” “substantially comprising,” and “essentially comprising,” which have established meanings in patent terminology.

[0049] Numerical ranges are described by their endpoints, including all integers and, where appropriate, fractions contained within the respective range, along with the described endpoints. This applies to numerical ranges, whether they are introduced by expressions such as "from…to…", "between…and…" or other expressions. Any numerical range described herein is intended to include all subranges contained therein.

[0050] As used herein, the terms “about” or “approximately”, when referring to measurable values ​​such as parameters, quantities, periods of time, etc., are intended to cover variations compared to a specified value, such as ±10% or less, preferably ±5% or less, more preferably ±1% or less, and even more preferably ±0.1% or less, provided that such variations are suitable for implementation in the disclosed invention. It should be understood that the values ​​referred to by the modifiers “about” or “approximately” are themselves specifically and preferably disclosed.

[0051] Furthermore, unless otherwise specified, the terms "first," "second," "third," etc., used in the specification and claims are used to distinguish similar elements and are not necessarily used to describe order or chronological sequence. It should be understood that these terms can be used interchangeably where appropriate, and embodiments of the invention described herein can operate in a different order than those described or illustrated herein.

[0052] The terms "one or more" or "at least one" are self-explanatory, referring to at least one member of one or more members or a group of members. Further exemplification, the term specifically includes referring to any one of the members, or any two or more of the members, such as any three or four, five or six or seven, etc., up to all members. In another instance, "one or more" or "at least one" can refer to 1, 2, 3, 4, 5, 6, 7 or more.

[0053] As used in this document, the term "and / or" when used in a list of two or more items means that any one of the listed items can be used alone, or any combination of two or more of the listed items can be used. For example, if a list is described as containing groups A, B, and / or C, then the list can contain a single A, a single B, a single C, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0054] The discussion of the background of the invention included herein is intended to explain the context of the invention. This should not be construed as an admission that any material mentioned was published, known, or common knowledge in any country prior to the priority date of any claim.

[0055] Throughout this disclosure, various publications, patents, and published patent specifications are cited by way of identifying reference. All documents cited in this specification are incorporated herein by reference in their entirety. In particular, the teachings or sections of such documents specifically mentioned herein are incorporated herein by reference.

[0056] Unless otherwise defined, all terms used in this disclosure, including technical and scientific terms, shall have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. To further guide the understanding of this invention, terminology definitions are included herein. When a particular term is defined in connection with a specific aspect or embodiment of the invention, its meaning or significance shall apply throughout this specification, and also to other aspects or embodiments of the invention, unless otherwise defined.

[0057] The following paragraphs define different aspects or embodiments of the invention in more detail. Unless explicitly stated otherwise, each aspect or embodiment so defined may be used in combination with any other aspect or embodiment. Specifically, any feature designated as preferred or advantageous may be used in combination with any other feature designated as preferred or advantageous.

[0058] Throughout this specification, the phrase "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of this aspect. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing in various places throughout the specification do not necessarily refer to the same embodiment, but may refer to the same embodiment. Furthermore, it will be apparent to those skilled in the art, based on this disclosure, that particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments. Moreover, while some embodiments described herein include some features included in other embodiments, they do not include all features included in other embodiments, and different combinations of features from different embodiments are intended to fall within the scope of this invention and form different embodiments, as understood by those skilled in the art. For example, in the appended claims, any claimed embodiment can be used in any combination.

[0059] As illustrated in the Experimental Section (which describes some representative embodiments of the invention), the inventors provide advantageous applications of certain neutrophil elastase (NE) binders (e.g., particularly human NE (hNE) binders and nucleic acid molecules, carriers containing such binders, viruses, and cells), as well as methods for manufacturing these substances and their use in medicine. Currently, anti-NE immunoglobulin monovariable domain reagents exhibit one or more advantages, such as acting as competitive inhibitors, demonstrating strong affinity, specificity, and inhibitory ability for NE. Furthermore, anti-NE ISVDs exhibit excellent stability in vivo, with a prolonged half-life in the lungs, especially when PEGylated, thus paving the way for therapeutic interventions and combination therapies involving anti-NE ISVDs.

[0060] Therefore, one aspect of the present invention provides a binder that binds to and inhibits neutrophil elastase (NE). In one embodiment, the binder competes with elastase inhibitor 3 (EI3) for binding to NE, and the binder comprises an immunoglobulin single variable domain (ISVD).

[0061] Another aspect of the invention provides a binder capable of specifically binding to and inhibiting neutrophil elastase (NE), wherein the binder comprises an immunoglobulin single variable domain (ISVD). In one embodiment, the binder competes with elastase inhibitor 3 (EI3) for binding to NE.

[0062] Therefore, another aspect of the present invention provides a binding agent comprising an immunoglobulin single variable domain (ISVD) that binds to at least one of the R36, A60, N61, P96, V97, S195, G218, and G219 residues of NE, preferably binding to two or more of the R36, A60, N61, P96, V97, S195, G218, and G219 residues, more preferably binding to three or more of the R36, A60, N61, P96, V97, S195, G218, and G219 residues of NE, and even more preferably binding to all of the R36, A60, N61, P96, V97, S195, G218, and G219 residues of NE, for example, binding to at least the R36, A60, N61, P96, V97, and S195 residues of NE.

[0063] Another aspect of the invention provides a binder comprising a structural element for specifically binding to and inhibiting neutrophil elastase (NE). Another aspect of the invention provides a binder comprising a structural element that specifically binds to and inhibits neutrophil elastase (NE) and competes with elastase inhibitor 3 (EI3) for NE binding. Another aspect of the invention provides a binder comprising a structural element that binds at least one of residues R36, A60, N61, P96, V97, S195, G218, and G219 of NE, preferably two or more of residues R36, A60, N61, P96, V97, S195, G218, and G219, more preferably three or more of residues R36, A60, N61, P96, V97, S195, G218, and G219, and even more preferably all of residues R36, A60, N61, P96, V97, S195, G218, and G219, for example, at least residues R36, A60, N61, P96, V97, and S195 of NE. Another aspect of the invention provides an antibody or antibody fragment (e.g., ISVD, especially VHH) comprising a structural element that specifically binds to and inhibits neutrophil elastase (NE). Yet another aspect of the invention provides an antibody or antibody fragment (e.g., ISVD, especially VHH) comprising a structural element that specifically binds to and inhibits neutrophil elastase (NE) and competes with elastase inhibitor 3 (EI3) for binding to NE. Another aspect of the invention provides an antibody or antibody fragment (e.g., ISVD, especially VHH) comprising a structural element that binds at least one of the residues R36, A60, N61, P96, V97, S195, G218, and G219 of NE, preferably two or more of the residues R36, A60, N61, P96, V97, S195, G218, and G219, more preferably three or more of the residues R36, A60, N61, P96, V97, S195, G218, and G219, and even more preferably all of the residues R36, A60, N61, P96, V97, S195, G218, and G219, for example, at least the residues R36, A60, N61, P96, V97, and S195 of NE.

[0064] Another aspect of the invention provides an anti-neutrophil elastase (NE) antibody or antibody fragment (e.g., ISVD, especially VHH) comprising a structural element that specifically binds to and inhibits neutrophil elastase (NE). Another aspect of the invention provides an anti-neutrophil elastase (NE) antibody or antibody fragment (e.g., ISVD, especially VHH) comprising a structural element that binds at least one of the residues R36, A60, N61, P96, V97, S195, G218, and G219 of NE, preferably two or more of the residues R36, A60, N61, P96, V97, S195, G218, and G219, more preferably three or more of the residues R36, A60, N61, P96, V97, S195, G218, and G219, and even more preferably all of the residues R36, A60, N61, P96, V97, S195, G218, and G219, for example, at least the residues R36, A60, N61, P96, V97, and S195 of NE.

[0065] Another aspect of the invention provides a composition comprising a binder (e.g., a pharmaceutical composition) that includes a structural element for specifically binding to and inhibiting neutrophil elastase (NE). Another aspect of the invention provides a composition comprising a binder (e.g., a pharmaceutical composition) that includes a structural element for specifically binding to and inhibiting neutrophil elastase (NE) and competing with elastase inhibitor 3 (EI3) for NE binding. Another aspect of the invention provides a composition (e.g., a pharmaceutical composition) comprising a binding agent containing a structural element that binds at least one of residues R36, A60, N61, P96, V97, S195, G218, and G219 of NE, preferably two or more of residues R36, A60, N61, P96, V97, S195, G218, and G219, more preferably three or more of residues R36, A60, N61, P96, V97, S195, G218, and G219, and even more preferably all of residues R36, A60, N61, P96, V97, S195, G218, and G219, for example, at least residues R36, A60, N61, P96, V97, and S195 of NE.

[0066] Another aspect of the invention provides a composition (e.g., a pharmaceutical composition) comprising an antibody or antibody fragment (e.g., ISVD, especially VHH) containing a structural element that specifically binds to and inhibits neutrophil elastase (NE). Another aspect of the invention provides a composition (e.g., a pharmaceutical composition) comprising an antibody or antibody fragment (e.g., ISVD, especially VHH) containing a structural element that specifically binds to and inhibits neutrophil elastase (NE) and competes with elastase inhibitor 3 (EI3) for binding to NE. Another aspect of the invention provides a composition (e.g., a pharmaceutical composition) comprising an antibody or antibody fragment (e.g., ISVD, especially VHH) containing a structural element that binds at least one of the residues R36, A60, N61, P96, V97, S195, G218, and G219 of NE, preferably two or more of the residues R36, A60, N61, P96, V97, S195, G218, and G219, more preferably three or more of the residues R36, A60, N61, P96, V97, S195, G218, and G219, and even more preferably all of the residues R36, A60, N61, P96, V97, S195, G218, and G219, for example, at least the residues R36, A60, N61, P96, V97, and S195 of NE.

[0067] Another aspect of the invention provides a composition (e.g., a pharmaceutical composition) comprising an anti-neutrophil elastase (NE) antibody or antibody fragment (e.g., ISVD, especially VHH), said antibody or antibody fragment comprising a structural element that specifically binds to and inhibits neutrophil elastase (NE). Another aspect of the invention provides a composition (e.g., a pharmaceutical composition) comprising an anti-neutrophil elastase (NE) antibody or antibody fragment (e.g., ISVD, especially VHH), said antibody or antibody fragment comprising a structural element that specifically binds to and inhibits neutrophil elastase (NE) and competes with elastase inhibitor 3 (EI3) for binding to NE. Another aspect of the invention provides a composition (e.g., a pharmaceutical composition) comprising an anti-neutrophil elastase (NE) antibody or antibody fragment (e.g., ISVD, especially VHH), said antibody or antibody fragment comprising a structural element that binds to at least one of NE residues R36, A60, N61, P96, V97, S195, G218, and G219, preferably binding to residues R36, A60, N61, or P96. Two or more of residues R36, A60, N61, P96, V97, S195, G218 and G219, more preferably three or more of residues R36, A60, N61, P96, V97, S195, G218 and G219, more preferably all of residues R36, A60, N61, P96, V97, S195, G218 and G219, for example, at least residues R36, A60, N61, P96, V97 and S195 of NE.

[0068] Another aspect of the invention provides a composition (e.g., a pharmaceutical composition) comprising a binder and an excipient (e.g., a pharmaceutical excipient), the binder comprising a structural element for specifically binding to and inhibiting neutrophil elastase (NE). Another aspect of the invention provides a composition (e.g., a pharmaceutical composition) comprising a binder and an excipient (e.g., a pharmaceutical excipient), the binder comprising a structural element for specifically binding to and inhibiting neutrophil elastase (NE) and competing with elastase inhibitor 3 (EI3) for NE binding. Another aspect of the invention provides a composition (e.g., a pharmaceutical composition) comprising a binder and an excipient (e.g., a pharmaceutical excipient), the binder comprising a structural element that binds at least one of residues R36, A60, N61, P96, V97, S195, G218, and G219 of NE, preferably two or more of residues R36, A60, N61, P96, V97, S195, G218, and G219, more preferably three or more of residues R36, A60, N61, P96, V97, S195, G218, and G219, and even more preferably all of residues R36, A60, N61, P96, V97, S195, G218, and G219, for example, at least residues R36, A60, N61, P96, V97, and S195 of NE.

[0069] Another aspect of the invention provides a composition (e.g., a pharmaceutical composition) comprising an antibody or antibody fragment (e.g., ISVD, especially VHH) and an excipient (e.g., a pharmaceutical excipient), wherein the antibody or antibody fragment contains a structural element that specifically binds to and inhibits neutrophil elastase (NE). Another aspect of the invention provides a composition (e.g., a pharmaceutical composition) comprising an antibody or antibody fragment (e.g., ISVD, especially VHH) and an excipient (e.g., a pharmaceutical excipient), wherein the antibody or antibody fragment contains a structural element that specifically binds to and inhibits neutrophil elastase (NE) and competes with elastase inhibitor 3 (EI3) for binding to NE. Another aspect of the invention provides a composition (e.g., a pharmaceutical composition) comprising an antibody or antibody fragment (e.g., ISVD, especially VHH) and an excipient (e.g., a pharmaceutical excipient), said antibody or antibody fragment comprising a structural element that binds to at least one of NE residues R36, A60, N61, P96, V97, S195, G218, and G219, preferably binding to residues R36, A60, N61, P96, ... Two or more of V97, S195, G218 and G219, more preferably three or more of residues R36, A60, N61, P96, V97, S195, G218 and G219, more preferably all of residues R36, A60, N61, P96, V97, S195, G218 and G219, for example, at least residues R36, A60, N61, P96, V97 and S195 of NE.

[0070] Another aspect of the invention provides a composition (e.g., a pharmaceutical composition) comprising an anti-neutrophil elastase (NE) antibody or antibody fragment (e.g., ISVD, especially VHH) and an excipient (e.g., a pharmaceutical excipient), said antibody or antibody fragment comprising a structural element that specifically binds to and inhibits neutrophil elastase (NE). Another aspect of the invention provides a composition (e.g., a pharmaceutical composition) comprising an anti-neutrophil elastase (NE) antibody or antibody fragment (e.g., ISVD, especially VHH) and an excipient (e.g., a pharmaceutical excipient), said antibody or antibody fragment comprising a structural element that specifically binds to and inhibits neutrophil elastase (NE) and competes with elastase inhibitor 3 (EI3) for binding to NE. Another aspect of the invention provides a composition (e.g., a pharmaceutical composition) comprising an anti-neutrophil elastase (NE) antibody or antibody fragment (e.g., ISVD, especially VHH) and an excipient (e.g., a pharmaceutical excipient), said antibody or antibody fragment comprising a structural element that binds to at least one of NE residues R36, A60, N61, P96, V97, S195, G218, and G219, preferably binding residues R36, A60, ... Two or more of N61, P96, V97, S195, G218 and G219, more preferably three or more of residues R36, A60, N61, P96, V97, S195, G218 and G219, more preferably all of residues R36, A60, N61, P96, V97, S195, G218 and G219, for example, at least residues R36, A60, N61, P96, V97 and S195 of NE.

[0071] The present invention also relates to uses and methods relating to the above-described binders, antibodies and compositions as described in other parts of this document.

[0072] Therefore, one aspect of the present invention provides a binder capable of specifically binding to and inhibiting neutrophil elastase (NE), wherein the binder comprises an immunoglobulin single variable domain (ISVD) comprising a complementarity-determining region 1 (CDR1) having the sequence shown in SEQ ID NO: 1 (GRTISLYR), a CDR2 having the sequence shown in SEQ ID NO: 2 (INWSGDMT), and a CDR3 having the sequence shown in SEQ ID NO: 3 (TADPKLLPLADSSYGY).

[0073] The term NE as used herein may specifically refer to human NE (hNE). The qualifier “human” used in connection with NE proteins herein may, in one interpretation, refer to the amino acid sequence of the NE protein. For example, NE proteins with the same amino acid sequence as those found in humans can be obtained through technical means, such as recombinant expression, cell-free translation, or non-biological peptide synthesis. Since current ISVDs aim to therapeutically target human NE, in another interpretation, the qualifier “human” may more specifically refer to NE proteins found or present in humans, regardless of whether the NE protein constitutes part of a human subject, organ, cell, or tissue, or whether it has been at least partially isolated from a human subject, organ, cell, or tissue. Those skilled in the art will understand that the amino acid sequence of a given natural protein (such as NE protein) may vary between or within different individuals of the same species due to normal genetic diversity (allelic variation, polymorphism) and / or due to differences in post-transcriptional or post-translational modifications. Any variants or isotypes of such natural proteins are included in references to or nomenclature of the protein.

[0074] Following further guidance and without any restrictions, human NE has been annotated in the National Center for Biotechnology Information (NCBI) GenBank (http: / / www.ncbi.nlm.nih.gov / ) under gene ID 1991. The amino acid sequence of the human wild-type NE precursor protein is annotated under GenBank accession number NP_001963.1, as follows: MTLGRRLACLFLACVLPALLLGGTALASEIVGGRRARPHAWPFMVSLQLRGGHFCGATLIAPNFVMSAAHCVANVNVRAVRVVLGAHNLSRREPTRQVFAVQRIFENGYDPVNLLNDIVILQLNGSATINANV QVAQLPAQGRRLGNGVQCLAMGWGLLGRNRGIASVLQELNVTVVTSLCRRSNVCTLVRGRQAGVCFGDSGSPLVCNGLIHGIASFVRGGCASGLYPDAFAPVAQFVNWIDSIIQRSEDNPCPHPRDPDPASRTH (SEQ ID NO: 13) Those skilled in the art will understand that any sequence represented in sequence databases or this application specification can be a precursor and may include portions removed during processing from mature molecules. Without limitation, the mature human NE sequence, after processing to remove the signal peptide, dipeptide SE, and C-terminal propeptide, can be as follows: IVGGRRARPHAWPFMVSLQLRGGHFCGATLIAPNFVMSAAHCVANVNVRAVRVVLGAHNLSREPTRQVFAVQRIFENGYDPVNLLNDIVILQLNGSATINANVQVAQLPA QGRRLGNGVQCLAMGWGLLGRNRGIASVLQELNVTVVTSLCRRSNVCTLVRGRQAGVCFGDSGSPLVCNGLIHGIASFVRGGCASGLYPDAFAPVAQFVNWIDSIIQ(SEQ ID NO:14) To further elucidate, and not only to extend, scientific knowledge regarding the biological relevance of NE, NE is a serine endopeptidase with a catalytic site composed of the amino acids Asp, His, and Ser. NE is the most abundant serine proteases found in neutrophils. It is primarily stored in high concentrations in the azurophilic granules of neutrophils. Upon neutrophil activation, NE can bind to chondroitin sulfate and heparan sulfate proteoglycans, thereby localizing to the cell surface. Due to the locally high concentration of NE on the neutrophil surface, which is temporarily higher than endogenous antiproteases, a proteolytic burst occurs on the neutrophil surface. While the proteolytic activity of NE is crucial for innate immune function, its release into the airway environment can lead to the progression of lung disease.

[0075] At a deeper level, norepinephrine (NE) upregulates the gene expression of mucin 5AC (MUC5AC), a major gel-forming mucin in respiratory tract secretions. NE can also activate intracellular signals, including reactive oxygen species (ROS), nicotinamide adenine dinucleotide phosphate (NADPH) quinone oxidoreductase 1 (NQO1), and epidermal growth factor receptor (EGFR), which contribute to the upregulation of MUC5AC expression. NE enhances epithelial permeability by releasing transforming growth factor α (TGFα) from the cell surface and by degrading connective proteins such as Zona occludins-1 and E-cadherin, thereby promoting the binding of TGFα-induced EGFR. In other words, NE allows the basolateral ligand TGFα to cross the cell apex and activate EGFR. Furthermore, NE can induce bronchial epithelial cells to secrete mucin, leading to airway mucus obstruction. In addition, NE induces goblet cell metaplasia, altering the composition of the airway epithelium and persistently increasing the production and secretion of mucin in the airways.

[0076] Within the airways, effective mucociliary clearance depends on the presence of sufficient mucin levels, adequate hydration of airway surface fluid, and healthy ciliated epithelium. However, norepinephrine (NE) disrupts every crucial component necessary for maintaining mucociliary clearance. In addition to increasing mucin abundance in the airways, NE also hinders airway surface fluid hydration by degrading cystic fibrosis transmembrane conductance regulator (CFTR) (a apical chloride channel) and activating ENaC (an apical epithelial sodium channel). Thus, the ionic regulation of airway hydration is disrupted. Furthermore, NE reduces ciliary motility and damages ciliary structure, further impairing the effectiveness of mucociliary clearance within the airways. Considering the role of NE in the airways, it becomes a suitable drug target for inflammatory lung diseases such as CF and COPD.

[0077] The anti-NE ISVD mentioned in this article inhibits NE by binding to at least a portion of the NE active site. By binding to the active site, anti-NE ISVD can block the contact between the substrate and serine proteases, thereby preventing proteolytic activity and interfering with the normal function of NE.

[0078] As is well known, hNE is a serine protease, meaning its catalytic activity depends on the serine residues present at its active site. The catalytic residue triplet in hNE consists of histidine 57 (H57), aspartic acid 102 (D102), and serine 195 (S195). Numbering follows the chymotrypsinogen scheme, and the correlation between this scheme and FASTA numbering is as follows... Figure 1 As shown.

[0079] Serine residues are important components of the catalytic site, responsible for nucleophilic attack during proteolysis. Histidine residues act as bases, deprotonating the serine hydroxyl group and thus enhancing its nucleophilicity. Aspartic acid residues (D102) stabilize histidine residues, helping to maintain the correct conformation of the active site.

[0080] The binding of a substrate or inhibitor to the enzyme's active site can be demonstrated by X-ray crystallography, as shown in Example 10, where the enzyme-substrate or enzyme-inhibitor complex is crystallized and then subjected to X-ray diffraction analysis. The resulting diffraction patterns can be used to determine the three-dimensional structure of the complex, including the binding interactions at the active site. Other methods include, but are not limited to, nuclear magnetic resonance (NMR) spectroscopy, directed mutagenesis of the active site, fluorescence spectroscopy, isothermal titration calorimetry (ITC), mass spectrometry, and enzyme kinetic experiments.

[0081] In one embodiment, anti-NE ISVD can inhibit NE activity, for example, in an in vitro assay using N-succinyl-Ala-Ala-Ala-p-nitroaniline substrate, its 50% inhibitory concentration (IC50) is [missing value]. 50The IC50 value is 60 nM or lower, preferably 50 nM or lower, more preferably 42 nM or lower; as determined in an in vitro assay using an elastin substrate, its IC50 value is... 50 The value is 20 to 70 nM, preferably 25 to 60 nM, more preferably 30 to 55 nM, even more preferably 35 to 50 nM, even more preferably 30 to 45 nM, even more preferably 35 to 40 nM, and most preferably 38.3 nM.

[0082] In another implementation, the anti-NE ISVD is able to bind to NE, whose dissociation constant (K) D The value is 4 nM or lower, preferably 3 nM or lower, as determined by biological layer interferometry (BLI).

[0083] However, in another embodiment, the Gibbs energy change (ΔG(H2O)) against NE ISVD is at least 25 kJ / mol, more preferably at least 30 kJ / mol, and even more preferably at least 35 kJ / mol, as measured by urea-induced development at 25°C and pH 7.

[0084] However, in another embodiment, the anti-NE ISVD includes a melting point (T m The temperature is at least 65°C, preferably at least 68°C, and more preferably at least 70°C.

[0085] In another embodiment, anti-NE ISVD can compete with α1-antitrypsin (AAT) for binding to NE.

[0086] In another embodiment, the anti-NE ISVD can bind to or within the NE region containing the NE active site. However, in another embodiment, the anti-NE ISVD can bind to the amino acid residues R36, A60, N61, P96, V97, S195, G218, and G219 of NE, such as... Figure 1 As shown.

[0087] In some embodiments, the anti-NE ISVD CDR3 can interact with amino acid residues R36, A60, N61, P96, S195, and V97 of NE according to the chymotrypsinogen numbering scheme. More specifically, amino acids P100, K101, D107, Y110, and Y112 in the anti-NE ISVD CDR3, numbered according to the FASTA sequence, can interact with amino acid residues R36, A60, N61, P96, V97, S195, G218, and G219 of NE according to the chymotrypsinogen numbering scheme. Residue P100 binds to S195 of the enzyme's catalytic triplet via a water molecule.

[0088] As used herein, the term "antibody" refers to antibodies in the broadest sense, generally meaning immunobinding agents. This term encompasses complete immunoglobulin molecules, immunoglobulin fragments with immunological effectiveness (i.e., fragments capable of specifically binding to antigens recognized by complete immunoglobulin molecules), constructs containing antigen-binding portions within modified immunoglobulin-like frameworks, and constructs containing antigen-binding portions within non-immunoglobulin-like frameworks or scaffolds. Antibody fragments include a portion of a complete antibody containing an antigen-binding region or its variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv, and single-domain sdFv (sdFv) antibodies, such as VL, VH, or VHH single-domain antibodies. Fusion proteins with heavy chain (VH) and light chain (VL) variable regions, often referred to as single-chain Fv (scFv), are also included in antibody fragments. Therefore, the term "antibody" includes, but is not limited to, intact monoclonal antibodies, intact polyclonal antibodies, multivalent (e.g., bivalent, trivalent, or higher) antibodies and / or multispecific (e.g., bispecific or higher specific) antibodies formed from at least two intact antibodies, and also includes any immunologically effective fragments of such antibodies, and multivalent and / or multispecific complexes of such fragments (e.g., biantibodies, triantibodies, tetraantibodies, multiantibodies). The term also includes, but is not limited to, intact antibodies and antibody fragments of non-human animal origin, and chimeric, humanized, or chimeric / humanized forms of such antibodies or antibody fragments, and further includes fully human antibodies or antibody fragments. More broadly, consideration is given to transplanting at least one complementarity-determining region (CDR) of an antibody from one source onto a framework from another source. The term "antibody" also includes any fusion protein, protein conjugate, or protein complex containing an immunoglobulin molecule or an immunologically effective fragment thereof, and chemically and / or enzymatically modified or derivatized immunoglobulin molecules or immunologically effective fragments thereof. The term "antibody" includes not only antibodies produced by methods involving immunization, but also any polypeptide containing at least one CDR capable of specifically binding to a homologous antigen epitope, whether such molecules are produced in vitro, in cell culture, or in vivo. For example, antibodies produced using recombinant DNA technology in cultured host cells (such as bacteria, yeast, or fungi, plant or animal cells) or non-human host organisms (such as transgenic plants or animals) are also included.

[0089] The aspects disclosed herein employ anti-NE immunoglobulin single variable domain (ISVD), more preferably anti-hNE ISVD. As used herein, the term "domain" (a domain of a polypeptide or protein) refers to a folded protein structure capable of maintaining its tertiary structure independently of the rest of the protein. Generally, domains confer independent functional properties on proteins and, in many cases, can be added to, removed from, or transferred to other proteins without losing the function of the rest of the protein and / or the domain itself. The term considered herein specifically refers to an "immunoglobulin domain," i.e., a globular region of an antibody chain (e.g., a chain of a conventional four-chain antibody or a heavy-chain antibody), or a polypeptide primarily composed of or composed of such globular regions. Structurally, immunoglobulin domains are described as retaining the immunoglobulin folding characteristics of antibody molecules, particularly comprising a bilayer sandwich structure consisting of approximately seven antiparallel β-chains arranged in two β-sheets, selectively stabilized by conserved disulfide bonds. The term "domain" as used herein specifically refers to an immunoglobulin variable domain.

[0090] The terms "single domain," "single variable domain," or "immunoglobulin single variable domain" define a molecule in which an antigen-binding site is present, and that the antigen-binding site is formed by a single immunoglobulin domain. This distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins or fragments thereof, in which two immunoglobulin domains, specifically two variable domains, interact to form an antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form the antigen-binding site. In this case, the complementarity-determining regions (CDRs) of the VH and VL will contribute to the antigen-binding site; that is, a total of six CDRs will be involved in the formation of the antigen-binding site. Therefore, the antigen-binding domains of conventional 4-chain antibodies (such as IgG, IgM, IgA, IgD, or IgE molecules) or Fab fragments, F(ab')2 fragments, Fv fragments (such as disulfide-linked Fv fragments), or scFv fragments, or biantibodies derived from such conventional 4-chain antibodies, are generally not considered immunoglobulin monovariable domains. This is because, in these cases, binding to the corresponding epitope of the antigen is usually not achieved through a single immunoglobulin domain, but rather through a pair of associated immunoglobulin domains (such as light chain and heavy chain variable domains), i.e., through the VH-VL pair of immunoglobulin domains co-binding the epitope of the corresponding antigen.

[0091] In contrast, immunoglobulin monovariable domains (MMUs) can specifically bind to antigen epitopes without pairing with other immunoglobulin variable domains. The binding site of an immunoglobulin MMU is formed by a single VH, VHH, or VL domain. Therefore, the antigen-binding site of an immunoglobulin MMU is formed by no more than three CDRs. An immunoglobulin MMU can be a light chain variable domain (VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain (VH-sequence or VHH-sequence) or a suitable fragment thereof; as long as it can form a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain, such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).

[0092] In a broad sense, ISVD is not limited to a specific biological source or a specific preparation method. The term "immunoglobulin single variable domain" encompasses variable domains from various sources, including mouse, rat, rabbit, donkey, human, shark (e.g., the so-called "IgNAR domain", see WO 05 / 18629 for example), and camelid variable domains.

[0093] Therefore, as envisioned herein, the operable antigen-binding mechanism of a single-domain antibody is based on a single variable domain of an immunoglobulin. In some preferred embodiments, this single domain may be a “heavy chain variable domain,” as used herein, meaning: (i) a variable domain derived from the heavy chain of a heavy chain antibody that naturally does not contain a light chain, including but not limited to heavy chain variable domains of camel or shark heavy chain antibodies; or (ii) a variable domain derived from the heavy chain of a conventional four-chain antibody (hereinafter also referred to as V). H This includes, but is not limited to, camelification of conventional four-chain antibody heavy chains (as further defined below) and variable domains (hereinafter also referred to as camelified V). H ), or any functional fragment thereof. In some preferred embodiments, the single domain may be as described in (i). Thus, in some embodiments, the single-domain antibody described herein is derived from a heavy chain antibody (V HH The heavy chain variable structural domain or its functional segment, i.e., its hNE binding segment. For example, the binding K of such segments with hNE. D K is not higher than the full-length reference sequence D The value should be 10 times that of the full-length reference sequence, preferably not higher than K. D Five times the value, more preferably not higher than K of the full-length reference sequence. D Twice the value, for example, its K D The value of K compared to the full-length reference sequence D The values ​​are essentially the same (e.g., + / - 1.5 times or + / - 1.2 times).

[0094] The “VHH domain,” also known as VHH, VHH antibody fragment, and VHH antibody, was originally described as the antigen-binding immunoglobulin (variable) domain of a “heavy chain antibody,” i.e., the antigen-binding immunoglobulin (variable) domain of a light chain antibody (Hamers-Casterman et al., Naturally occurring antibodies devoid of light chains. Nature 1993, vol. 363, 446-448). The term “VHH domain” was chosen to distinguish these variable domains from the heavy chain variable domains present in conventional 4-chain antibodies (which are conventionally and herein referred to as “VH domains”) and the light chain variable domains present in conventional 4-chain antibodies (which are conventionally and herein referred to as “VL domains”). Thus, in some preferred embodiments, a single domain may be a VHH domain, or in other words, an anti-NE antibody may be a VHH single-domain antibody, or simply a VHH antibody.

[0095] In some preferred embodiments, the anti-NE antibody may be a nanobody. The term "nanobody" (Nb) as used herein ("Nanobody®", "Nanobodies®" and "Nanoclone®" are registered trademarks of Ablynx NV, Ghent, Belgium) refers to a single variable domain derived from a naturally occurring heavy chain antibody (excluding the light chain), particularly antibodies found in camels (Hamers-Casterman et al., 1993, above; Desmyter et al., Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme. Nat Struct Biol. 1996, vol. 3, 803-811), and is therefore often referred to as VHH antibody or VHH. Camels include Old World camels (Bactrian camel (…) Camelus bactrianus ) and dromedary camels ( Camelus dromedarius And New World camel species (such as alpacas) Vicugna pacos ), llamas Lama glama ), Australian camel ( Lama guanicoe ) and Vicunian alpacas ( Vicugna vicugnaThe term nanobody as used herein is not limited in its broadest sense to a specific biological source or a specific method of preparation. For example, nanobody in its broadest sense may include an immunobinding agent obtained by: (1) isolating the VHH domain of a naturally occurring heavy chain antibody; (2) expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3) “humanizing” a naturally occurring VHH domain or expressing a nucleic acid encoding such a humanized VHH domain; (4) “camelizing” a naturally occurring VH domain from any animal species (particularly mammalian species, such as humans) or expressing a nucleic acid encoding such a camelized VH domain; (5) “camelizing” a “domain antibody” or “Dab” as described in the art or expressing a nucleic acid encoding such a camelized VH domain; (6) preparing a protein, polypeptide or other amino acid sequence using known synthetic or semi-synthetic techniques; (7) preparing a nucleic acid encoding a nanobody using known nucleic acid synthesis techniques and then expressing the nucleic acid obtained therefrom; and / or (8) any combination of one or more of the above.

[0096] For further description of VHH and nanobodies, see Muyldermans' review article (Reviews in Molecular Biotechnology 74: 277-302, 2001), and the following patent applications, which are mentioned as general background art: Vrije Universiteit Brussel's WO94 / 04678, WO95 / 04079, and WO96 / 34103; Unilever's WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231, and WO 02 / 48193; and Vlaams Instituut voor Biotechnologie (VIB)'s WO 97 / 49805, WO WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO03 / 055527; WO 03 / 050531 of Algonomics NV and Ablynx NV; WO01 / 90190 of the National Research Council of Canada; WO 03 / 025020 (= EP 1433793) of the Antibody Institute; and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825, and other published patent applications by Ablynx NV.

[0097] Mentions of single-domain antibodies (including VHH and domain antibodies) also include their functional fragments, which retain at least some or all of the functional activity and / or binding specificity of the original immunoglobulin single variable domain (e.g., the VHH domain) from which these fragments originate. The length and / or size of the functional fragments are not particularly limited and may include, but are not limited to, forms representing the deletion or truncation of the N-terminus and / or C-terminus of the original immunoglobulin single variable domain. For example, these fragments may represent at least about 50% (in terms of amino acid number) of the continuous amino acid sequence of said original immunoglobulin single variable domain, for example, at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%. This term encompasses fragments generated by any mechanism, such as, but not limited to, heterologous expression of truncated forms of immunoglobulin monovariable domains, or fragments generated by physical, chemical, or enzymatic proteolytic processes. Typically, functional fragments of immunoglobulin monovariable domains (such as VHH domains) disclosed herein contain at least a number of amino acid residues that constitute at least one complementarity-determining region of the original immunoglobulin monovariable domain (such as the VHH domain) from which they originate.

[0098] The antibodies considered in this document are anti-NE antibodies, i.e., antibodies that specifically bind to NE. The term "specific binding" as used throughout this specification means a binding agent to one or more target molecules or analytes that substantially excludes binding to other random or unrelated molecules, and optionally substantially excludes binding to other structurally related molecules. In other words, an antibody is said to specifically bind to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0099] Antibody binding occurs primarily with epitopes on NE proteins. The term "epitaph" encompasses any polypeptide determinant capable of specifically binding to immunoglobulins or T-cell receptors. Epitope determinants may include chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and may possess specific three-dimensional structural features and / or specific charge characteristics. An epitope is a region on an antigen that binds to an antibody. Antibody specifically binds to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0100] The term "specificity" refers to the number of different types of antigens or antigenic determinants that a particular antigen-binding molecule or antigen-binding protein (such as an antibody) can bind to. The specificity of an antigen-binding protein can be determined based on affinity and / or avidity. Affinity is determined by the equilibrium constant (Ka) of the dissociation of the antigen from the antigen-binding protein. D K represents an indicator that measures the binding strength between antigenic determinants and antigen-binding sites on antigen-binding proteins. D The smaller the value, the stronger the binding strength between the antigen determinant and the antigen-binding molecule (or, affinity can also be expressed as the affinity constant (K)). A ), that is, 1 / K D It will be apparent to those skilled in the art that affinity can be determined by known methods, depending on the specific antigen of interest. Affinity is a measure of the strength of binding between an antigen-binding molecule (such as an antibody) and its associated antigen. Affinity is related to the affinity between the antigenic determinant and its antigen-binding site on the antigen-binding molecule, as well as the number of associated binding sites present on the antigen-binding molecule. Typically, the dissociation constant (K0) of the antigen-binding protein (e.g., antibody) is a key factor in determining affinity. D ) is 1x10 -5 Up to 1x10 -12 mol / L (M) or lower, preferably 1x10 -7 Up to 1x10 -12 M or lower, preferably 1x10 -8 Up to 1x10 -12 M or lower, or even better, 1x10 -9 Up to 1x10 -12 M or lower, for example, 1x10 -9 Up to 1x10 -10 M, or 1x10 -10 Up to 1x10 -11 M, where K D = [AB][AG] / AB-AG], where AB represents antibody, AG represents antigen, and AB-AG represents antibody-antigen complex. Any value greater than 10 -4 M of K D The K value is generally considered to indicate nonspecific binding. Preferably, the K value indicates that the antibody binds to the target antigen. D Less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, for example less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM, for example about 500 pM, about 600 pM, about 700 pM, about 800 pM, or about 900 pM. In some preferred examples, the Kc of the antibody binding to the target antigen is... DValues ​​range from 500 pM to 3 nM. The specific binding of antigen-binding proteins to antigens or antigenic determinants can be determined by any known suitable method, including, for example, Scatchard diagram analysis and / or competitive binding assays such as immunoassays, enzyme immunoassays (EIA) and sandwich competitive assays, as well as various variants known in the art.

[0101] In some particularly preferred embodiments, the anti-NE ISVD may comprise, consist primarily of, or be composed of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 4.

[0102] SEQ ID NO: 4 includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 1, 2, and 3, which are indicated by underline, bold, and underline-bold fonts, respectively. The aforementioned CDR sequences are annotated in SEQ ID NO: 4 according to their IMGT numbers. In some embodiments, the anti-NE ISVD may comprise an amino acid sequence or a functional fragment thereof having a sequence identity with SEQ ID NO: 4 preferably at least 91%, at least 92%, at least 93%, or at least 94%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, of SEQ ID NO: 4, or is primarily composed of or constitutes thereof.

[0103] In a specific implementation, the anti-NE ISVD comprises CDR1, CDR2, and CDR3 sequences, each present in SEQ ID NO: 4, annotated according to any of the numbering systems IMGT, Kabat, Chlotia, Martin, or Aho. These corresponding CDR1-3 annotations and their corresponding frame regions (FR1-4) are shown in [the diagram / image / image]. Figure 2 On the SEQ ID NO:4 sequence.

[0104] In some particularly preferred embodiments, the anti-NE ISVD comprises, is primarily composed of, or is composed of the amino acid sequence shown in SEQ ID NO: 4. In some particularly preferred embodiments, the anti-NE ISVD is NbE201 as described in the examples.

[0105] SEQ ID NO:4 contains FR1, FR2, FR3 and FR4 as shown in SEQ ID NO:17, 18, 19 and 20, which are indicated in bold in SEQ ID NO:4 in the following order:

[0106] The aforementioned FR sequence is annotated in SEQ ID NO:4 according to the IMGT number. In some embodiments, anti-NEISVD may independently comprise frame region 1 (FR1), frame region 2 (FR2), frame region 3 (FR3), and frame region 4 (FR4), wherein frame region 1 (FR1) has a sequence identity with the sequence shown in SEQ ID NO:17 (QVQLQESGGGLVQAGGSLRLSCVVP) having at least 90%, preferably at least 91%, at least 92%, at least 93%, or at least 94%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; frame region 2 (FR2) has a sequence identity with the sequence shown in SEQ ID NO:18 (MGWFRQAPGKEREFVAG) having at least 90%, preferably at least 91%, at least 92%, at least 93%, or at least 94%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and frame region 3 (FR3) has a sequence identity with the sequence shown in SEQ ID NO:17 (QVQLQESGGGLVQAGGSLRLSCVVP). The sequence shown in NO: 19 (DYVDSVKGRFTISRDNAKNTVYLEMNSLKPEDTAIYYC) has at least 90%, preferably at least 91%, at least 92%, at least 93%, or at least 94%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and the frame region 4 (FR4) has a sequence identity with the sequence shown in SEQ ID NO: 20 (WGQGTQVTVSS) having at least 90%, preferably at least 91%, at least 92%, at least 93%, or at least 94%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0107] In some embodiments, the anti-NE ISVD may comprise FR1-4 sequences that independently have at least 90%, preferably at least 91%, at least 92%, at least 93%, or at least 94%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the corresponding FR1-4 sequence present in SEQ ID NO: 4, said FR1-4 sequences being numbered according to any one of the numbering systems IMGT, Kabat, Chlotia, Martin, or Aho.

[0108] The term "protein" generally refers to a large molecule consisting of one or more polypeptide chains. The term "polypeptide" generally refers to a linear polymer chain of amino acid residues linked by peptide bonds. A "peptide bond," "peptide link," or "amide bond" is a covalent bond formed between two amino acids, releasing a water molecule when the carboxyl group of one amino acid reacts with the amino group of another. The terms "protein" and "polypeptide" are used interchangeably, especially when a protein consists of only one polypeptide chain. These terms are not limited to any minimum length of polypeptide chain. Polypeptide chains consisting primarily of or less than 50 (≤ 50) amino acids, such as those consisting primarily of or less than 45, ≤ 40, ≤ 35, ≤ 30, ≤ 25, ≤ 20, ≤ 15, ≤ 10, or ≤ 5 amino acids, are generally referred to as "peptides." In the context of proteins, polypeptides, or peptides, "sequence" refers to the arrangement of amino acids in a chain from the amino terminus to the carboxyl terminus, where adjacent residues in the sequence are continuous in the primary structure of the protein, polypeptide, or peptide. These terms can encompass proteins, polypeptides, or peptides that are naturally occurring, recombinant, semi-synthetic, or synthetically produced. Thus, for example, proteins, polypeptides, or peptides may exist in or be isolated from nature, for example, naturally or endogenously produced or expressed by cells or tissues and selectively isolated therefrom; or proteins, polypeptides, or peptides may be recombinant, i.e., produced by recombinant DNA technology, and / or may be partially or wholly synthesized by chemical or biochemical means. Without limitation, proteins, polypeptides, or peptides may be recombinantly produced and selectively isolated from suitable host or host cell expression systems (e.g., suitable bacterial, yeast, fungal, plant, or animal host or host cell expression systems), or recombinantly produced through cell-free translation or cell-free transcription and translation, or produced through non-biological peptide, polypeptide, or protein synthesis. The term also encompasses proteins, polypeptides, or peptides that carry one or more co-expressed or post-expression-type modifications, such as, but not limited to, glycosylation, esterification, acetylation, amidation, phosphorylation, sulfonation, methylation, PEGylation (polyethylene glycol is typically covalently linked to the N-terminus or one or more Lys residues on the side chain), ubiquitination, SUMOylation, cysteine ​​modification, glutathioneization, methionine oxidation to methionine sulfoxide or methionine sulfone, signal peptide removal, N-terminal Met removal, conversion of proenzymes or prohormones to their active forms, etc. Such co-expressed or post-expression-type modifications can be introduced in vivo by host cells expressing these proteins, polypeptides, or peptides (the co-translational or post-translational protein modification mechanism can be native to the host cell and / or the host cell can be genetically engineered to include one or more (additional) co-translational or post-translational protein modification functions), or can be introduced in vitro by chemical (e.g., PEGylation) and / or biochemical (e.g., enzymatic) modifications to isolated proteins, polypeptides, or peptides.

[0109] In some embodiments, the anti-NE ISVD binder may comprise a polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety may be linked to the ISVD, for example, specifically, it may be covalently linked to the ISVD, for example, specifically, an amino acid covalently linked to the ISVD. The ISVD may contain one or more amino acids suitable for linking the PEG moiety thereto, for example, through genetic engineering.

[0110] In one implementation, amino acid residues, such as lysine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, or preferably cysteine, can be introduced into the C-terminus or N-terminus of the anti-NE ISVD via genetic engineering. This modification can functionalize the anti-NE ISVD by linking a PEG moiety, thereby prolonging its half-life in the lungs. Site-specific PEGylation of the anti-NE ISVD with amino acid residues prevents its aggregation during nebulization and spray drying, whereas unmodified anti-NE ISVD readily aggregates under similar conditions, as illustrated in the following examples.

[0111] In a preferred embodiment, a linker is introduced via genetic engineering prior to the C-terminal cysteine ​​residue of the anti-NE ISVD. This functionalizes the anti-NE ISVD by linking the polyethylene glycol (PEG) moiety, thereby extending its half-life in the lungs. Site-specific PEGylation of the anti-NE ISVD via the linker near the cysteine ​​residue prevents its aggregation during nebulization and spray drying, whereas unmodified anti-NE ISVD readily aggregates under similar conditions, as illustrated in the following examples.

[0112] In certain embodiments, any linker may be a peptide or polypeptide linker of one or more amino acids. In some embodiments, all linkers in the molecule may be peptide or polypeptide linkers. More specifically, peptide linkers may be 1 to 20 amino acids in length, preferably 1 to 15 amino acids, and more preferably 12 to 14 amino acids. For example, the length of the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, preferably 12, 13, or 14 amino acids. The properties of the amino acids constituting the linker are not particularly relevant, as long as the biological activity of the molecule stretches to which the linker is attached is not substantially impaired. Preferably, the linker is substantially non-immunogenic and / or not easily cleaved by proteolytic enzymes. In some embodiments, the linker may contain a predicted secondary structure, such as an α-helix. However, linkers predicted to exhibit a flexible, random helical structure are preferred. Linkers with a tendency to form β-chains may not be preferred or should be avoided. Because cysteine ​​residues can form intermolecular disulfide bonds, they may not be preferred or should be avoided. Basic or acidic amino acid residues, such as arginine, lysine, histidine, aspartic acid, and glutamic acid, may not be preferred or should be avoided due to their ability to generate unintended electrostatic interactions. In some preferred embodiments, the peptide linker may comprise, consist primarily of, or consist of the following amino acids: glycine, serine, alanine, phenylalanine, threonine, proline, and combinations thereof, including their D-isomers and analogs. In some preferred embodiments, the peptide linker may comprise, consist primarily of, or consist of the following amino acids: glycine, serine, alanine, threonine, proline, and combinations thereof, including their D-isomers and analogs. In even more preferred embodiments, the peptide linker may comprise, consist primarily of, or consist of the following amino acids: serine, threonine, proline, and combinations thereof, including their D-isomers and analogs. In some embodiments, the peptide linker may consist only of serine, threonine, and proline residues. In some embodiments, the peptide linker may consist of only serine, or only threonine, or only proline or its D-isomer or analogue. In some embodiments, the linker may contain serine, threonine, and proline residues, wherein the ratio of proline to serine is from 7:1 to 1:7 (by amount), for example, a proline:serine ratio of about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 3:2, about 1:1, about 1:2, about 2:3, about 1:3, about 1:4, about 1:5, or about 1:6.Preferably, proline can be more abundant than serine, for example, the proline:serine ratio is 7:4, 3:2, or 2:1 (by quantity), and preferably, the proline:serine ratio is 3:2 (by quantity). In some embodiments, the linker may comprise serine, threonine, and proline residues, wherein the ratio of threonine to proline residues is from 7:1 to 1:7 (by quantity), for example, the proline:threonine ratio is about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, or about 1:6. Preferably, proline can be more abundant than threonine, for example, the proline:threonine ratio is 7:3, 3:1, or 2:1 (by quantity), and preferably, the proline:threonine ratio is 2:1 (by quantity). In some embodiments, the linker may comprise serine, threonine, and proline residues, wherein the ratio of serine to threonine is 6:1 to 1:6 (by amount), for example, a serine:threonine ratio of about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, or about 1:6. Preferably, serine may be more abundant than threonine, for example, a serine:threonine ratio of 2:1 to 4:3 (by amount), preferably a serine:threonine ratio of 4:3 (by amount). In a preferred embodiment, the ratio of serine, threonine, and proline residues may be 4:3:7. In some embodiments, both the N-terminal and C-terminal residues of the linker are serine residues; or both the N-terminal and C-terminal residues of the linker are proline residues; or the N-terminal residue is a serine residue and the C-terminal residue is a proline residue; or the N-terminal residue is a proline residue and the C-terminal residue is a threonine residue. In some embodiments, the peptide linker may consist only of proline residues or their D-isomers or analogs, preferably only of proline residues. By way of example, but not limited thereto, the peptide linker referred to herein may comprise, consist primarily of, or consist of the amino acid sequence defined by SEQ ID NO 16: SPSTPPTPSPSTPP.

[0113] Polyethylene glycol (PEG) is a polymeric compound containing repeating ethylene glycol units, also known as polyethylene oxide (PEO) or polyethylene oxide (POE). The PEGs considered in this paper consist of linear ethylene oxide oligomers or polymer chains, and the use of PEGs with branched, Y-shaped, or multi-arm geometries is considered. PEG has a wide range of average molecular weights, for example, the range of average molecular weights, especially number average molecular weights (Mw) is: 190-210 Da (PEG 200), about 285-315 Da (PEG 300), about 380-420 Da (PEG 400), about 570-630 Da (PEG 600), about 855-900 Da (PEG 900), about 950-1050 Da (PEG 1000), about 1900-2200 Da (PEG 2000), about 2700-3300 Da (PEG 3000), about 3500-4500 Da (PEG 4000) or about 7000-9000 Da (PEG 8000).

[0114] PEG can be linked to cysteine ​​residues by forming thioester bonds. Typically, PEG reactants containing chemical groups are used, such as maleimide and its derivatives, like N-ethylmaleimide (NEM) and N-(2-aminoethyl)maleimide (AEM); haloacetamides and vinyl sulfone groups, which can react with the thiol groups of cysteine ​​to form thioether bonds. Cysteine ​​is an amino acid with a thiol group (-SH) on its side chain. When a PEG reactant containing a chemical group reacts with cysteine, a covalent bond is formed, causing the PEG molecule to attach to the cysteine ​​residue.

[0115] As described above, PEG reactants containing chemical groups can have a wide range of average molecular weights, for example, having the following average molecular weight ranges: 1 to 100 kDa, about 1 to 80 kDa, about 1 to 70 kDa, about 1 to 60 kDa, about 1 to 50 kDa, about 1 to 40 kDa, about 1 to 30 kDa, about 1 to 20 kDa, about 1 to 10 kDa, preferably having an average molecular weight of 10 kDa, 20 kDa, or 40 kDa.

[0116] Furthermore, PEGylation protects anti-NE ISVD from protease degradation. The PEGylated form of anti-NE ISVD exhibits a significantly increased half-life in the lungs. PEGylation prolongs the drug's half-life, thereby reducing the frequency of treatment.

[0117] Its advantage is that the anti-NE ISVD is stable for at least 3 months at 4°C or even up to 37°C without experiencing a decrease in activity. This makes the formulation suitable for storage and transportation without freezing, for example, it can be stored for a long time under refrigeration (about 4°C) or without refrigeration at ambient temperature (about 22 to 25°C).

[0118] The term "amino acid" encompasses naturally occurring amino acids, naturally encoded amino acids, non-naturally encoded amino acids, non-naturally occurring amino acids, amino acid analogs, and amino acid mimics that function in a manner similar to naturally occurring amino acids. All of these amino acids exist as D- and L-stereoisomers, provided their structures allow for such stereoisomerism. In this document, amino acids are referred to by their names, by commonly known three-letter symbols, or by single-letter symbols recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature. "Naturally encoded amino acid" refers to one of the 20 most common amino acids, or pyrrolysine, pyrrolinocarboxylysine, or selenocysteine. The 20 common amino acids are: alanine (A or Ala), cysteine ​​(C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), and tyrosine (Y or Tyr). "Non-naturally encoded amino acids" refer to amino acids that are not among the 20 common amino acids, nor are they pyrrolidone, pyrrolinoline carboxylysine, or selenocysteine. The term includes, but is not limited to, amino acids produced by modifying naturally encoded amino acids (e.g., post-translational modifications), rather than naturally encoded amino acids themselves being incorporated into the growing polypeptide chain by the translation complex, such as, but not limited to, N-acetylglucosamine-L-serine, N-acetylglucosamine-L-threonine, and O-phosphotyrosine. Other examples of non-naturally encoded amino acids, non-natural amino acids, or modified amino acids include: 2-aminohexanoic acid, 3-aminohexanoic acid, β-alanine, β-aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, piperidine acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoserine, homocysteine, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyllysine, N-methylvaline, orovaline, oroleucine, or ornithine. In addition, it includes amino acid analogs, in which one or more individual atoms are replaced by different atoms, isotopes of the same atom, or different functional groups.In addition, this includes non-natural amino acids and amino acid analogues described in Ellman et al., Methods Enzymol. 1991, vol. 202, 301-36. Incorporating non-natural amino acids into proteins, polypeptides, or peptides may have advantages in many ways. For example, proteins, polypeptides, or peptides containing D-amino acids exhibit greater stability in vitro or in vivo compared to those containing L-amino acids. More specifically, proteins, polypeptides, or peptides containing D-amino acids may exhibit greater resistance to endogenous peptidases and proteases, thereby improving the bioavailability of the molecule and extending its lifespan in vivo.

[0119] In terms of amino acid sequences, the term "sequence identity" refers to the degree of overall sequence identity between amino acid sequences read from the N-terminus to the C-terminus (i.e., including all amino acid sequences described in the comparison), expressed as a percentage. Sequence alignment can be performed using suitable algorithms, and sequence identity can be determined using known methods. ， To determine sequence identity. Exemplary but not limiting algorithms include those based on the Basic Local Alignment Search Tool (BLAST), originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10), such as the “Blast 2 Sequence” algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250), for example, using published default settings or other suitable settings (e.g., for the BLASTN algorithm: cost of opening the gap = 5, cost of expanding the gap = 2, penalty for mismatch = -2, reward for matching = 1, gap x_dropoff = 50, expected value = 10.0, word length = 28; or for the BLASTP algorithm: matrix = Blosum62 (Henikoff et al., 1992, Proc. Natl. Acad. Sci., 89: 10915-10919), cost of opening the gap = 11, cost of expanding the gap = 1. Expected value = 10.0, word length = 3).

[0120] An example procedure for determining the percentage of identity between a specific amino acid sequence and a query amino acid sequence (e.g., an anti-NE ISVD sequence, an anti-NE VHH sequence, or a CDR sequence) is to use the Blast 2 sequence (Bl2seq) algorithm to align each read of the two amino acid sequences from the N-terminus to the C-terminus. This algorithm is available as a web application or a standalone executable (BLAST version 2.2.31+) on the NCBI website (www.ncbi.nlm.nih.gov) with appropriate algorithm parameters. Examples of appropriate algorithm parameters include: matrix = Blosum62, cost of opening a gap = 11, cost of expanding a gap = 1, expectation = 10.0, word length = 3. If the two compared sequences are identical, the output will display these identical regions as the aligned sequence. If the two sequences are not identical, the output will not display the aligned sequence. After alignment, the number of matches is determined by counting the positions of identical amino acid residues in the two sequences. The identity percentage is determined as follows: divide the number of matches by the length of the query sequence, and then multiply the result by 100. The percentage identity value can (but not necessarily) be rounded to one decimal place. For example, 78.11, 78.12, 78.13, and 78.14 can be rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 can be rounded up to 78.2. Furthermore, it is worth noting that the detailed view of each aligned segment in the Bl2seq output conveniently includes the identity percentage.

[0121] When an amino acid sequence differs, varies, or is different from another amino acid sequence—for example, when the preceding amino acid sequence shares a certain degree or percentage of sequence identity with the following amino acid sequence, or when the preceding amino acid sequence differs from the following amino acid sequence by a certain number of amino acids—this sequence variation may consist of the addition of one or more amino acids (e.g., the addition of one amino acid or an extension of two or more consecutive amino acids at one position or at two or more positions in the amino acid sequence), deletion (e.g., the deletion of one amino acid or an extension of two or more consecutive amino acids at one position or at two or more positions in the amino acid sequence), and / or substitution (e.g., the substitution of one amino acid or an extension of two or more consecutive amino acids at one position or at two or more positions in the amino acid sequence with an extension of one amino acid or an extension of two or more consecutive amino acids).

[0122] Preferably, the substitution of one or more amino acids, particularly one or more single amino acid substitutions, can be conservative amino acid substitutions. Conservative amino acid substitution refers to the substitution of one amino acid for another with similar properties. Conservative amino acid substitutions include substitutions within the following group: valine, alanine, and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. Nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (i.e., basic) amino acids include arginine, lysine, and histidine. Negatively charged (i.e., acidic) amino acids include aspartic acid and glutamic acid. Substituting one member of the same group for another member of the aforementioned polar, basic, or acidic groups can be considered a conservative substitution. In contrast, nonconservative substitution refers to replacing one amino acid with another amino acid that has different characteristics.

[0123] On the other hand, a nucleic acid molecule is provided that contains a polynucleotide sequence encoding the binding agent described herein, or a vector containing the nucleic acid molecule is provided, or an expression cassette or expression vector as defined herein is provided.

[0124] On the other hand, it relates to expression cassettes or expression vectors that contain a nucleic acid molecule as defined herein and a promoter operatively linked to that nucleic acid molecule. Preferably, the expression cassette or expression vector can be configured to efficiently express a nucleic acid molecule encoding anti-NE ISVD or a functionally active variant or fragment thereof in host cells. For example, in bacterial cells, fungal cells (including yeast cells), animal cells, or mammalian cells (including human cells and non-human mammalian cells). Preferably, the expression cassette or expression vector is configured to efficiently express a nucleic acid molecule encoding anti-NE ISVD or a functionally active variant or fragment thereof.

[0125] “Encoding” specifically refers to the correspondence between a nucleic acid sequence or a portion thereof in a template-transcriptional product (e.g., RNA or RNA analog) relationship and another nucleic acid sequence, or the correspondence between the genetic code of an organism and a specific amino acid sequence (e.g., the amino acid sequence of one or more target proteins or polypeptides).

[0126] As used herein, the term "expression vector" or "vector" refers to a nucleic acid molecule, typically DNA, into which a nucleic acid fragment (preferably a recombinant nucleic acid molecule as defined herein) can be inserted and cloned (i.e., amplified). Thus, a vector typically contains one or more unique restriction sites and is capable of autonomous replication in a specific host cell or vector organism, thereby enabling the replication of the cloned sequence. The vector may also preferably contain selection markers, such as antibiotic resistance genes, to select recipient cells containing the vector. Vectors may include, but are not limited to, plasmids, phage particles, bacteriophages, phage-derived vectors, PACs, BACs, linear nucleic acids (e.g., linear DNA), viral vectors, etc., as appropriate (see, for example, Sambrook et al., 1989; Ausubel, 1992). Expression vectors are typically configured to allow and / or enable the expression of the nucleic acid or ORF introduced therein in a target expression system, such as in vitro, in host cells, host organs, and / or in a host organism. For example, expression vectors may advantageously contain suitable regulatory sequences.

[0127] Key factors in selecting a specific vector include: the selection of the recipient host cell, which can easily identify and select recipient cells containing the vector from recipient cells that do not contain the vector; the required number of vector copies in the specific recipient cell; whether it is desired for the vector to integrate into the chromosome of the recipient cell or to remain outside the chromosome; and whether it is desired to be able to "shuttle" the vector between recipient cells of different species.

[0128] Expression vectors can be autonomous or integrated. Recombinant nucleic acids can be introduced into host cells in the form of expression vectors, such as plasmids, bacteriophages, transposons, granules, or viral particles. Recombinant nucleic acids can be maintained extrachromosomally or integrated into the cell's chromosomal DNA. Expression vectors can contain selection marker genes that encode proteins required for cell survival under selected conditions (e.g., URA3, which encodes an enzyme required for uracil biosynthesis; or TRP1, which encodes an enzyme required for tryptophan biosynthesis), allowing for the detection and / or selection of cells transformed with the target nucleic acid. Expression vectors can also contain autonomously replicating sequences (ARS).

[0129] An integrative vector typically comprises a tandem arrangement of at least a first insertable DNA fragment, a selectable marker gene, and a second insertable DNA fragment. Each of the first and second insertable DNA fragments is about 200 nucleotides in length (e.g., about 250, 300, 350, 400, 450, 500, or 1000 or more) and has a nucleotide sequence homologous to a portion of the genomic DNA of the host cell species to be transformed. A nucleotide sequence containing the target gene to be expressed is inserted between the first and second insertable DNA fragments of the vector, either before or after the marker gene. The integrative vector may be linearized prior to transformation to facilitate the integration of the target nucleotide sequence into the host cell genome.

[0130] As used herein, the term "promoter" refers to a DNA sequence capable of inducing transcription of a gene. A promoter can be recognized by RNA polymerase, which then initiates transcription. Therefore, a promoter contains a DNA sequence that binds directly to RNA polymerase or participates in the recruitment of RNA polymerase. The promoter sequence may also contain "enhancer regions," which are one or more DNA regions that can bind to proteins (i.e., trans-acting factors) to enhance the transcriptional level of genes within a gene cluster. Enhancers are typically located at the 5' end of a coding region, but can also be located separately from the promoter sequence, for example, within an intron region of a gene or at the 3' end of a gene's coding region. Preferred promoters include, but are not limited to, promoters known to be expressed in bacterial cells. Some preferred but non-restrictive promoters that can be used with these host cells include: the lac promoter (and its derivatives, such as the lacUV5 promoter) for expression in Escherichia coli; arabinose promoters; left-hand and right-hand promoters for λ phage; promoters for the tryptophan (trp) operon; heterozygous lac / trp promoters (tac); T7 promoters and other T phage promoters; promoters for the Tn10 tetracycline resistance gene; and engineered variants of the above promoters.

[0131] "Operationally operable connections" refer to connections between regulatory sequences and sequences to be expressed in a manner that allows for that expression. For example, sequences (e.g., promoters and ORFs) may be considered operablely connected if the connection between them does not have the following characteristics: (1) leading to the introduction of frameshift mutations, (2) interfering with the promoter's ability to direct ORF transcription, and (3) interfering with the ORF's ability to transcribe from the promoter sequence. Therefore, "operationally operable connections" may mean integration into a genetic construct that allows expression regulatory sequences (e.g., promoters) to effectively control the expression of target coding sequences (e.g., nucleic acid molecules as defined herein).

[0132] Promoters can be constitutive or inducible (conditional) promoters. A constitutive promoter should be understood as one that expresses constant values ​​under standard culture conditions. An inducible promoter is one that responds to one or more inducible signals. For example, inducible promoters can be regulated chemically (e.g., their transcriptional activity is regulated by the presence or absence of chemical inducers such as alcohols, tetracyclines, steroids, metals, or other small molecules) or physically (e.g., their transcriptional activity is regulated by the presence or absence of physical inducers such as light, high temperature, or low temperature). Inducible promoters can also be indirectly regulated by one or more transcription factors, which themselves are directly regulated by chemical or physical signals.

[0133] Before introducing the vector into the target cells, it can be placed in bacterial cells such as Escherichia coli (E. coli). E. coli The vector is grown (e.g., amplified) in the bacterial environment. The vector DNA can be isolated from bacterial cells using any method known in the art, which enables the purification of the vector DNA from the bacterial environment. The purified vector DNA can be extracted in large quantities using phenol, chloroform, and ether to ensure the absence of *E. coli* proteins in the plasmid DNA preparation, as these proteins are toxic to mammalian cells.

[0134] It should be understood that any genetic engineering modifications described herein can also be conditional. For example, a gene can be conditionally deleted using a site-specific DNA recombinase (e.g., the Cre-loxP system) (see, for example, Gossen et al., 2002, Ann. Rev. Genetics, 36: 153-173 and US 20060014264).

[0135] Expression vectors or expression cassettes may also contain one or more selection markers that encode proteins required for cell survival under selected conditions, allowing for the detection and / or selection of cells transformed with a target nucleic acid, including one or more genes required to produce leucine (e.g., LEU2), uracil (e.g., URA3), adenine (e.g., ADE2), lysine (e.g., LYS5), arginine, tryptophan or glycerol utilization (Gut), and hygromycin B phosphotransferase (hph) markers. Some preferred but non-limiting examples of such selection markers are genes that provide resistance to antibiotics (e.g., ampicillin, chloramphenicol, tetracycline, or kanamycin); genes that provide resistance to temperature; or genes that allow host cells or host organisms to be maintained in a culture medium lacking certain factors, compounds, and / or (food) components essential for the survival of untransformed cells or organisms.

[0136] Expression vectors or expression cassettes can be integrated into the genome of a host cell. For example, expression vectors or expression cassettes can contain zeta elements, such as long terminal repeat sequences of retrotransposons, such as, but not limited to, Ylt1 or Tyl6 retrotransposons or other retrotransposons known to those skilled in the art. In one embodiment of the invention, the integration is targeted integration.

[0137] Alternatively, the expression vector or expression cassette may be replicated rather than integrated. For example, a replicated expression vector or expression cassette may contain one or more autosomal replication sequences (ARS). The ARS may contain a centromere (CEN) and an origin of replication (ORI). For example, the ARS may be ARS18 or ARS68.

[0138] The antibodies described herein can be formulated into pharmaceutical compositions. Therefore, any mention of the use of antibodies in treatment (or any variation thereof) also covers such uses of pharmaceutical compositions containing antibodies.

[0139] In some embodiments, the pharmaceutical composition may comprise anti-NE ISVD, a nucleic acid molecule or carrier encoding anti-NE ISVD, or cells or viruses containing anti-NE ISVD, as well as pharmaceutically acceptable carriers, diluents, and / or excipients. In a preferred embodiment, the pharmaceutical composition comprises other inhibitors of NE.

[0140] The terms “pharmaceutical composition” and “pharmaceutical formulation” are used interchangeably. The pharmaceutical compositions described herein contain one or more pharmaceutically acceptable carriers in addition to one or more active ingredients (e.g., antibodies). Suitable pharmaceutical excipients depend on the dosage form and type of the active ingredient and can be selected by those skilled in the art (e.g., see Handbook of Pharmaceutical Excipients, 7th Edition, 2012, edited by Rowe et al.).

[0141] The terms “carrier” or “excipient” used herein are used interchangeably and broadly include any and all solvents, diluents, buffers (e.g., neutral buffered salt solutions, phosphate buffered salt solutions, or optionally Tris-HCl, acetate, or phosphate buffers), surfactants (e.g., Tween® 80, polysorbate 80), protein stabilizers (e.g., sugars, polyols, polymers, cyclodextrins), colloids, dispersion media, carriers, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavorings, aromas, thickeners, agents for achieving sustained release, coating agents, antifungal agents, and preservatives (e.g., Thimerosal). TMMedia and reagents such as benzyl alcohol, antioxidants (e.g., ascorbic acid, sodium metabisulfite), tension control agents, absorption delay agents, adjuvants, and fillers (e.g., lactose, mannitol) are well known in the art for their use in the formulation of pharmaceutical compositions. Acceptable diluents, carriers, and excipients generally do not adversely affect the recipient's homeostasis (e.g., electrolyte balance). The use of such media and reagents in pharmaceutical active substances is well known in the art. Such substances should be non-toxic and should not interfere with the activity of the active ingredient. Acceptable carriers may include biocompatible, inert, or bioabsorbable salts, buffers, oligosaccharides or polysaccharides, polymers, viscosity modifiers, preservatives, etc. An exemplary carrier is physiological saline (0.15 M NaCl, pH 7.0 to 7.4). Another exemplary carrier is 50 mM sodium phosphate and 100 mM sodium chloride.

[0142] The specific properties of the carrier or other substance will depend on the route of administration. For example, the pharmaceutical composition may be in the form of a parenteral acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Preferably, the pH of the pharmaceutical formulation is within the physiological pH range, for example, particularly, the pH of the formulation is between about 5 and about 9.5, more preferably between about 6 and about 8.5, and even more preferably between about 7 and about 7.5.

[0143] The pharmaceutical composition can be administered systemically or locally. It can be formulated to suit parenteral and / or non-parenteral administration. Specific routes of administration include subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, intrathecal, oral, rectal, buccal, local, nasal, ocular, intra-articular, intra-articular, subarachnoid, bronchial, lymphatic, vaginal, and uterine administration.

[0144] Administration can be a periodic injection of the drug composition via bolus, or continuous or uninterrupted administration via intravenous or intraperitoneal injection from an external (e.g., IV bag) or internal (e.g., biodegradable implant, bioartificial organ, or implanted host cell population) reservoir. Administration of the drug composition can be achieved using suitable delivery methods, such as: pumps, microencapsulation, sustained-release polymer implants, macroencapsulation, injection (subcutaneous, intravenous, intra-arterial, intramuscular, or other suitable sites), or oral administration as capsules, liquids, tablets, pills, or sustained-release formulations.

[0145] Examples of parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, pump delivery, encapsulated cell delivery, liposome delivery, needle-based injection, needle-free injection, nebulizers, aerosol generators, electroporation, and transdermal patches.

[0146] In some embodiments, the pharmaceutical compositions described herein can be formulated for parenteral administration. Preferably, the pharmaceutical compositions described herein can be formulated for nasal or pulmonary administration, or administered using suitable devices such as nebulizers, dry powder inhalers, or metered-dose inhalers.

[0147] In a specific implementation, the device comprises a pharmaceutical composition and may be a nebulizer, a metered-dose inhaler, or a dry powder inhaler.

[0148] The quantity and properties of the pharmaceutical composition, as well as the duration of administration for a single dose, depend on the delivery system chosen. Various devices are known in the art for administering anti-NE ISVDs by inhalation into the lungs of a patient requiring such treatment. These devices include metered-dose inhalers, liquid nebulizers, dry powder inhalers, nebulizers, vaporizers, and similar devices. When using certain suitable delivery systems (such as nebulizers), the frequency of administration and duration of activation will vary depending on the concentration of anti-NE ISVDs in the aerosol powder. Higher concentrations of inhaled dry powder in the nebulizer solution allow for shorter administration periods. Similarly, metered-dose inhalers can produce higher aerosol concentrations, resulting in shorter operating times to reach the desired powder dose. On the other hand, devices such as dry powder inhalers can deliver anti-NE ISVDs until a certain amount of anti-NE ISVDs is expelled. In this type of inhaler, the amount of anti-NE ISVDs in a given amount of powder determines the dose administered in a single dose.

[0149] Pharmaceutical compositions containing anti-NE ISVD may optionally include additives, such as fillers, carriers, or excipients. Additives may be included in dry powders to dilute the powder to meet the delivery requirements of a specific powder inhaler, facilitate the processing of the pharmaceutical composition, impart favorable powder properties to the pharmaceutical composition, promote powder dispersion from the inhalation device, stabilize the pharmaceutical composition (e.g., antioxidants or buffers), impart flavor to the pharmaceutical composition, etc. Typical additives include monosaccharides, disaccharides, and polysaccharides; cellulose, cellulose derivatives, sugar alcohols (such as sorbitol, lactose, glucose, raffinose, metriose, lactitol, maltitol, trehalose, sucrose, and mannitol), starch, polyvinylpyrrolidone, lipids, and lipid excipients (such as phosphatidylcholine or lecithin); and amino acids (such as arginine, glycine, and leucine), etc.

[0150] Inhalation of the pharmaceutical composition of the present invention to the respiratory tract advantageously allows for local delivery of the formulation, thereby maximizing the effect of anti-NE ISVD in the respiratory tract. Furthermore, local application of the pharmaceutical composition to the respiratory tract via inhalation prevents dilution of the administered dose of anti-NE ISVD in systemic circulation, thus significantly improving the treatment ratio.

[0151] Those skilled in the art will recognize that the above description is merely illustrative and not exhaustive. In fact, many other formulation technologies, pharmaceutically acceptable excipients, and carrier solutions are well known to those skilled in the art, as are the development of suitable dosages and treatment regimens for the use of the specific compositions described herein in a variety of administration or treatment regimens.

[0152] The antibody dosage or administration described herein may optionally be combined with one or more other active compounds, depending on the specific circumstances, and should generally be adjusted accordingly to achieve the best effect. Therefore, the unit dose and regimen depend on the nature and severity of the disease to be treated, as well as on factors such as: the subject's species, sex, age, weight, general health condition, diet, method and timing of administration, immune status, individual responsiveness of the person or animal to be treated, the efficacy, metabolic stability and duration of action of the compound used, and whether the treatment is acute, chronic or prophylactic, or whether other active compounds have been administered in addition to the formulation of this invention. Unrestricted, depending on the type and severity of the disease, the typical dose of the molecules described herein (e.g., typical daily dose or typical intermittent dose, such as every two days, every three days, every four days, every five days, every six days, weekly, every 1.5 weeks, every two weeks, every three weeks, monthly, or other typical doses) may, based on the factors described above, range from about 10 µg / kg to about 100 mg / kg of subject weight, for example, from about 100 µg / kg to about 100 mg / kg of subject weight, or from about 200 µg / kg to about 75 mg / kg of subject weight, or from about 500 µg / kg to about 50 mg / kg of subject weight, or from about 1 mg / kg to about 25 mg / kg of subject weight, or from about 1 mg / kg to about 10 mg / kg of subject weight, for example, each dose may be about 100 µg / kg, about 200 µg / kg, about 300 µg / kg, about 400 µg / kg, etc. µg / kg, approximately 500 µg / kg, approximately 600 µg / kg, approximately 700 µg / kg, approximately 800 µg / kg, approximately 900 µg / kg, approximately 1.0 mg / kg, approximately 2.0 mg / kg, approximately 5.0 mg / kg, approximately 10 mg / kg, approximately 15 mg / kg, approximately 20 mg / kg, approximately 30 mg / kg, approximately 40 mg / kg, approximately 50 mg / kg, approximately 75 mg / kg, or approximately 100 mg / kg of subject weight.

[0153] For certain lung diseases, such as COPD and CF, there are multiple treatment options, including antibiotics, bronchodilators, and anti-inflammatory drugs. In lung diseases associated with norepinephrine (NE) activity, antibiotics are commonly used to treat and prevent bacterial infections. Targeting bacteria helps reduce NE release, thereby reducing damage to the lungs. In CF, a new class of drugs is also known that are designed to specifically target the defective cystic fibrosis transmembrane conductance regulator (CFTR) protein that causes CF. These drugs work by correcting the dysfunction of the CFTR protein, thereby improving the function of chloride ion channels and restoring the balance of ion transport in affected cells. Examples of CFTR regulators include ivacaftor, lumacaftor / ivacaftor, tezacaftor / ivacaftor, and elexacaftor / tezacaftor / ivacaftor. Ivacalito is used to treat certain CFTR gene mutations, while lumamacalito / ivacalito, terzacalito / ivacalito, and itracapto / terzacalito / ivacalito are specific combinations for treating CFTR gene mutations. In some implementations, anti-NE ISVDs may be used in combination with antibiotics, bronchodilators, and anti-inflammatory drugs as a treatment for NE-targeting lung diseases. In one specific implementation, anti-NE ISVDs are combined with CFTR modulators to treat subjects with lung diseases such as CF, including ivacalito, lumamacalito / ivacalito, terzacalito / ivacalito, and itracapto / terzacalito / ivacalito.

[0154] On the other hand, it involves a kit, such as a diagnostic kit, which contains a binder, a nucleic acid molecule or vector, a cell or a virus.

[0155] On the other hand, it involves the binders, nucleic acid molecules or carriers, cells or viruses or pharmaceutical compositions described herein, used in medicine.

[0156] Some implementation schemes involve binders, nucleic acid molecules or carriers, cells or viruses or pharmaceutical compositions for the prevention or treatment of inflammatory diseases in subjects.

[0157] The term “prevention,” or its alternative forms such as “avoidance” or “prevention,” generally refers to preventing something from happening, appearing, or existing, or delaying the occurrence or onset of something, particularly through one or more preventive (preventive) measures. For example, current uses or methods may be applied to subjects who have not yet exhibited symptoms of one or more inflammatory diseases and may prevent, for example, the occurrence or appearance of said one or more inflammatory disease symptoms within a given period of time.

[0158] As used throughout this specification, the terms "therapy" or "treatment" refer to the reduction or measurable reduction of one or more symptoms or pathological conditions (such as disease or patient). Measurable reduction includes a statistically significant decrease in any measurable symptom or marker. Generally, these terms include both curative treatment and treatments designed to reduce symptoms and / or delay disease progression. These terms include both therapeutic treatment of an existing pathological condition and preventative or preventive measures aimed at preventing or reducing the chance of the pathological condition occurring. Beneficial or desired clinical outcomes include, but are not limited to: disease prevention, reduction of disease incidence, relief of disease-related symptoms, reduction of disease severity, stabilization of disease, delay or slowing of disease progression, improvement or relief of disease, or combinations thereof. In some embodiments, these terms may be associated with therapeutic treatment. In some other embodiments, these terms may be associated with preventative treatment. Treatment performed during remission of a chronic pathological condition may also be considered as constituting therapeutic treatment. The term may cover in vitro or in vivo treatment as required by the context of this invention.

[0159] The term "subject" as used herein generally and preferably refers to a human being, but may also include non-human animals, preferably warm-blooded animals, and even more preferably mammals, such as non-human primates, rodents, canines, felines, equines, sheep, pigs, etc. The term "non-human animal" includes all vertebrates, such as mammals, including non-human primates (especially higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and non-mammals such as chickens, amphibians, reptiles, etc. In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human mammal. In some preferred embodiments of the methods and uses taught herein, the subject is a human subject. In other embodiments, the subject is an experimental animal or animal substitute used as a disease model. This term does not refer to a specific age or sex. Therefore, adults, newborns, or fetuses (regardless of sex) should be included in the scope of subjects.

[0160] One embodiment relates to a binder, nucleic acid molecule, carrier, cell or virus or pharmaceutical composition for use, wherein the inflammatory disease is a lung disease, preferably selected from mucoid obstructive pulmonary diseases (such as CF, COPD, bronchiectasis, ciliary dyskinesia and acute respiratory distress syndrome) and inflammatory bowel disease.

[0161] As used herein, the term "inflammatory disease" generally includes all diseases and conditions associated with inflammation, or in other words, all diseases and conditions containing an inflammatory component. Such inflammatory diseases can include acute inflammation (e.g., inflammation caused by injury or infection) or chronic inflammation. Examples of inflammatory diseases, exemplarily but not limitingly, are inflammatory lung diseases (e.g., mucoid obstructive lung diseases such as CF, COPD, emphysema, pulmonary fibrosis, bronchiectasis, ciliary dyskinesia, and acute respiratory distress syndrome; and non-mucoid obstructive lung diseases such as pulmonary hypertension), respiratory allergic diseases (e.g., asthma, allergic rhinitis, allergic lung disease), pulmonary autoimmune diseases, tuberculosis, inflammatory nasal and sinus diseases (e.g., chronic sinusitis), inflammatory bowel disease, Crohn's disease, ulcerative colitis, and inflammatory skin diseases (e.g., hidradenitis suppurativa).

[0162] Another aspect of the invention provides binders, nucleic acid molecules or carriers, cells or viruses or kits for diagnosing inflammatory diseases in subjects.

[0163] A further aspect involves cells or viruses that contain nucleic acid molecules or vectors, optionally wherein the cells are capable of expressing a binding agent or expressing a binding agent, or the virus is configured to induce the expression of a binding agent in recipient cells infected with the virus.

[0164] Cells (host cells) suitable for small-scale or large-scale production of anti-NE ISVD include, but are not limited to, prokaryotic or eukaryotic cells. For example, prokaryotes such as bacterial cells can be used. The host can be, for example, actinomycetes, such as Streptomyces, such as *Streptomyces azurite*. S. coelicolor For example, *Streptomyces azureense* A3(2), (strains M145 or M600), *Streptomyces purplish-blue* ( S. lividans ), Streptomyces cinnamon S. cinnamoneous ), or Escherichia coli. Other examples may include other Gram-positive bacteria, such as bacteria of the genus Actinomycetes, such as bacteria of the genus Mycobacterium, such as the pathogenic Mycobacterium tuberculosis (Mycobacterium tuberculosis). Mycobacterium tuberculosis ), Mycobacterium bovis ( M. bovis ), African mycobacterium ( M. africanum ) and Micromycobacteria ( M. microti ); Mycobacterium leprae ( M. leprae Another example of Gram-positive bacteria is the genus Clostridium, which includes pathogenic bacteria such as Clostridium difficile (Clostridium difficile). Clostridium difficile Clostridium botulinum (a human pathogen) C. botulinum Clostridium perfringens ( C. perfingens ) and Clostridium tetani ( C. tetani ), and bacteria with potential industrial applications, such as Clostridium acetobutyricum (Clostaphylococcus aureus). C. acetylbutylictum), Clostridium thermophilum ( C. thermocellum ) and Clostridium londauricum ( C. ljungdahlii Other Gram-positive bacterial genera may include *Bacillus*, *Listeria*, *Staphylococcus*, *Clostridium*, *Corynebacterium*, *Streptococcus*, and *Enterococcus*. Host cells can also be Gram-negative bacteria, including Enterobacteriaceae, which includes human pathogens such as Salmonella and *Escherichia coli*. Other examples of Gram-negative bacterial genera include *Pseudomonas*, *Bordezoella*, *Borrelia*, *Brucella*, *Campylobacter*, *Francis*, *Haemophilus*, *Klebsiella*, *Neisseria*, *Proteus*, *Rickettsia*, *Vibrio*, *Yersinia*, *Moraxella*, *Helicobacter*, *Stenotrophomonas maltophilia*, *Bdellovibrio*, acetic acid bacteria, Legionella, cyanobacteria, spirochetes, green sulfur bacteria, and green non-sulfur bacteria, etc. Important Gram-negative pathogens include cocci, which cause sexually transmitted diseases (Neisseria gonorrhoeae). Neisseria gonorrhoeae )), meningitis (Neisseria meningitidis ( Neisseria meningitidis )) and respiratory symptoms (Moraxella catarrhalis) Moraxella catarrhalis Other medically significant Gram-negative bacilli include a variety of species. Some of these bacteria primarily cause respiratory problems (e.g., Haemophilus influenzae). Hemophilus influenzae ), Klebsiella pneumoniae ( Klebsiella pneumoniae Legionella pneumophila ( Legionella pneumophila ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa Some of these bacteria primarily cause urinary system problems (E. coli). Escherichia coli ), Proteus mirabilis ( Proteus mirabilis Enterobacter cloacae () Enterobacter cloacae Serratia marcescens ( ) Serratia marcescens Some of these bacteria primarily cause gastrointestinal problems (Helicobacter pylori). Helicobacter pylori Salmonella enteritidis () Salmonella enteritidis ), Salmonella typhi Salmonella typhi The host cell may also include eukaryotes, such as yeast (examples of which may include: yeasts used for industrial production, such as the genus *Saccharomyces* (e.g., *Saccharomyces* var. *saccharomyces*). S. cerevisiae )), genus *Schizozymes* (such as *Schizozymes* ( S. pombe ) and methyltrophic yeasts of the genus Pichia [e.g., Pichia pastoris ( P. pastoris )] and Candida spp., as well as Hansenula polymorpha ( Hansenula polymorpha ); pathogenic yeasts, such as Candida species [e.g., Candida albicans ( C. albicans ) and tropical Candida ( C. tropicalis )], and Cryptococcus genus [such as Cryptococcus neoformans ( C. neoformansFungi (which may include: pathogenic fungi, such as Candida, Aspergillus [e.g., Aspergillus fumigatus ( A. fumigatus ) and aflatoxin ( A. flavus Cryptococcus, Histoplasma, such as Histoplasma capsulatum ( H. capsulatum Pneumocystis jirovecii genus [e.g., Pneumocystis jirovecii (Pneumocystis jirovecii)], Pneumocystis carinii genus [e.g., Pneumocystis carinii (Pneumocystis carinii)] P. jirovecii )] and the genus *Botrytis* [such as *Botrytis cinerea* ( S. chartarum Fungi used in industrial production, such as Aspergillus spp. [especially Aspergillus niger ( )]; fungi used in industrial production, such as Aspergillus spp. [especially Aspergillus niger ( )] A. niger ) and Aspergillus oryzae ( A. oryzae )]) and members of the genus Neurospora, such as Neurospora crassa ( N. crassa Plant cells and mammalian cells, avian cells, whether in cell culture or within a tissue portion. Bacterial host expression systems (such as *E. coli*) or yeast expression systems (such as *Pichia pastoris*). Pichia pastoris )) could be a better option.

[0165] As used herein, the term "virus" may be used interchangeably with the terms "host virus" or "genetically engineered host virus," which includes a recombinant nucleic acid molecule or vector encoding anti-NE ISVD according to any different embodiment of the invention. The virus can be introduced into a suitable host cell or host organism, which allows for the expression and, preferably, the secretion of anti-NE ISVD.

[0166] Another aspect of the present invention provides a method for determining the level of active neutrophil elastase in a sample, the method comprising contacting the sample with a binding agent and detecting at least the neutrophil elastase bound to the binding agent.

[0167] Anti-NE ISVD can be used to determine the level of active neutrophil elastase in specimens. Both AAT and anti-NE ISVD bind to the active site of NE. The binding of AAT to NE plays a crucial role by inhibiting the proteolytic activity of NE, preventing excessive protein breakdown. AAT achieves this inhibition by irreversibly binding to NE within its active site, thereby blocking its proteolytic function. Once AAT forms a complex with NE, NE cannot engage in further interactions, including binding to anti-NE ISVD.

[0168] Therefore, anti-NE ISVD cannot bind to NE molecules that have formed a complex with AAT or optionally with other endogenous inhibitors, such as Elafin, secretory leukocyte protease inhibitors (SLPIs), α1-antichymotrypsin (ACT), α2-macroglobulin, or monocyte neutrophil elastase inhibitors (MNEIs or SerpinB1). Conversely, anti-NE ISVD can only bind to active NE molecules that have not formed a complex with AAT or optionally with other endogenous inhibitors. NbE201 recognizes and binds to unoccupied active sites of NE, thereby modulating NE activity. Therefore, the binding and inhibition of anti-NE ISVD depends on the availability of active NE molecules, as NEs that have formed a complex with AAT cannot be contacted by anti-NE ISVD. Thus, anti-NE ISVD can be used in methods for determining the level of active neutrophil elastase by detecting the level of anti-NE ISVD bound to NE, wherein the level of anti-NE ISVD that has formed a complex with NE indicates the level of active NE that has not formed a complex in the sample. This can provide information about the level of NE activity in the sample.

[0169] Another aspect relates to a method for diagnosing, prognosing, and / or monitoring inflammatory diseases (optionally any inflammatory diseases described herein) in subjects, the method comprising: - The biological sample obtained from the subject will be contacted with the binder, and - The level and / or activity of neutrophil elastase in a sample are determined by detecting at least the amount of neutrophil elastase bound to the binder.

[0170] The term "contact" as used in this article also includes "exposure" and "interaction," generally referring to the act of bringing a binder into physical or functional contact with a sample or test substance. When a binder contacts a sample, it means that the binder is brought into direct or indirect contact with the sample under conditions generally favorable to antibody-antigen binding, with the aim of detecting, recognizing, or binding a specific antigen present in the sample. More specifically, when a binder contacts a sample, it means that the binder can contact active neutrophil elastase with the aim of detecting, recognizing, or binding active NE present in the sample.

[0171] In certain methods for detecting levels of active NE proteins, the binding agents described herein may be used. Affinity-based assays, such as immunological assays, include, but are not limited to, immunohistochemistry, immunocytochemistry, flow cytometry, mass flow cytometry, fluorescence-activated cell sorting (FACS), fluorescence microscopy, fluorescence cell sorting using microfluidic systems, techniques based on (immuno)affinity adsorption, such as affinity chromatography, magnetic particle separation, magnetically activated cell sorting, or bead-based cell sorting using microfluidic systems, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA) and ELISPOT-based techniques, radioimmunoassay (RIA), Western blotting, etc.

[0172] In further examples, such methods may include mass spectrometry analysis methods. Generally, any mass spectrometry (MS) technique that can obtain precise information about the peptide mass, and preferably also obtain fragments and / or (partial) amino acid sequences of the selected peptide (e.g., tandem mass spectrometry, MS / MS; or post-source decay, TOF MS), can be used to separate, detect, and / or quantify markers (e.g., preferably peptides, polypeptides, or proteins). Suitable peptide MS and MS / MS techniques and systems are well known (e.g., see Methods in Molecular Biology, vol. 146: “Mass Spectrometry of Proteins and Peptides”, by Chapman, ed., Humana Press 2000, ISBN 089603609x; Biemann 1990. Methods Enzymol 193: 455-79; or Methods in Enzymology, vol. 402: “Biological Mass Spectrometry”, by Burlingame, ed., Academic Press 2005, ISBN 9780121828073), and can be used in this paper. MS devices, instruments, and systems suitable for the analysis of biomarker peptides may include, but are not limited to: matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) MS; MALDI-TOF post-source decay (PSD); MALDI-TOF / TOF; surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF) MS; electrospray ionization mass spectrometry (ESI-MS); ESI-MS / MS; ESI-MS / (MS) n(n is a positive integer); ESI 3D or linear (2D) ion trap MS; ESI triple quadrupole MS; ESI quadrupole orthogonal TOF (Q-TOF); ESI Fourier transform MS system; Silicon-based desorption / ionization (DIOS); Secondary ion mass spectrometry (SIMS); Atmospheric pressure chemical ionization mass spectrometry (APCI-MS); APCI-MS / MS; APCI-(MS) n Atmospheric pressure photoionization mass spectrometry (APPI-MS); APPI-MS / MS; and APPI-(MS) n Peptide ion fragmentation in a tandem MS (MS / MS) apparatus can be achieved using methods established in the art, such as collision-induced dissociation (CID). Multiple reaction monitoring (MRM) can be employed using mass spectrometry detection and quantitative markers, as described, for example, in Kuhn et al., 2004 (Proteomics 4: 1175-86). MS peptide analysis methods can be advantageously combined with upstream peptide or protein separation or fractionation methods (e.g., chromatography and other methods).

[0173] In other examples, such methods may include chromatography. The term "chromatography" encompasses methods for separating substances, such as chemical or biological substances, such as markers, preferably peptides, polypeptides, or proteins, which are referred to in the art as such substances and are widely available. In preferred methods, chromatography refers to the separation of a mixture of substances ("analytes") carried by a moving liquid or gas stream ("mobile phase") into individual components due to the differential distribution of the analytes between the mobile phase and the stationary phase as the analytes flow around or over a stationary liquid or solid phase ("stationary phase"). The stationary phase can typically be a finely divided solid, filter paper, a liquid film on a solid surface, etc. Chromatography is also widely applicable to the separation of biologically derived chemical compounds, such as amino acids, proteins, protein fragments, or peptides.

[0174] Chromatographic methods are preferably column chromatography (i.e., in which a stationary phase is deposited or packed into a column), preferably liquid chromatography, and more preferably HPLC. Specific details of chromatographic methods are well known in the art, and for further guidance, see, for example, Meyer M., 1998, ISBN: 047198373X, and “Practical HPLC Methodology and Applications”, Bidlingmeyer, BA, John Wiley & Sons Inc., 1993. Exemplary types of chromatographic methods include, but are not limited to: high-performance liquid chromatography (HPLC), normal-phase HPLC (NP-HPLC), reversed-phase HPLC (RP-HPLC), ion-exchange chromatography (IEC) (such as cation-exchange chromatography or anion-exchange chromatography), hydrophilic-interaction chromatography (HILIC), hydrophobic-interaction chromatography (HIC), size exclusion chromatography (SEC) (including gel filtration chromatography or gel permeation chromatography), chromatographic focusing, affinity chromatography (such as immunoaffinity chromatography, immobilized metal affinity chromatography), etc.

[0175] Other techniques for separating, detecting, and / or quantifying biomarkers (e.g., peptides, polypeptides, or proteins) may also be used, and optionally in combination with any of the analytical methods described above. These methods include, but are not limited to, chemical extraction partitioning, isoelectric focusing (IEF) (including capillary isoelectric focusing (CIEF), capillary isovelocity electrophoresis (CITP), capillary electrochromatography (CEC), etc.), one-dimensional polyacrylamide gel electrophoresis (PAGE), two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), capillary gel electrophoresis (CGE), capillary zone electrophoresis (CZE), micelle electrokinetic chromatography (MEKC), free-flow electrophoresis (FFE), etc.

[0176] In some embodiments, reagents as described herein (e.g., binding agents (e.g., oligonucleotide primers)) may contain detectable labels. The term "label" refers to any atom, molecule, part, or biomolecule that can be used to provide detectable (preferably quantifiable) readings or properties and can be attached to or become part of a target entity, such as a binding agent. Labels can be appropriately detected, for example, by mass spectrometry, spectroscopy, optical methods, colorimetry, magnetic methods, photochemical methods, biochemical methods, immunochemical methods, or chemical methods. Labels include, but are not limited to, dyes; radioactive labels, such as... 32 P, 33 P, 35 S, 125 I, 131I; electron-dense reagents; enzymes (e.g., horseradish peroxidase or alkaline phosphatase commonly used in immunoassays); binding moieties, such as biotin-streptavidin; haptens, such as digoxigenin; luminescent, phosphorescent, or fluorescent moieties; quality tags; and fluorescent dyes alone or in combination with moieties whose emission spectra can be inhibited or altered by fluorescence resonance energy transfer (FRET).

[0177] In some embodiments, the binder may be tagged to allow detection with another reagent (e.g., a probe binding chaperone). For example, such a tag could be biotin, streptavidin, a His tag, a MyC tag, maltose, maltose-binding protein, or any other type of tag with a binding chaperone known in the art. Examples of bindings that can be used in a probe:binding chaperone array can be any binding, such as biotin:streptavidin, His tag:metal ion (e.g., Ni...). 2+ Maltose, maltose-binding protein, etc.

[0178] Protein-binding conjugates can bind or connect to detection reagents for convenient detection. Examples of detection reagents include, but are not limited to: luminescent labels; colorimetric labels, such as dyes; fluorescent labels; or chemical labels, such as electroactive agents (e.g., ferrocyanide); enzymes; radioactive labels; or radio frequency labels. Detection reagents can be particles. Examples of such particles include, but are not limited to: colloidal gold particles; colloidal sulfur particles; colloidal selenium particles; colloidal barium sulfate particles; colloidal ferric sulfate particles; metal iodate particles; silver halide particles; silica particles; colloidal metal (hydrated) oxide particles; colloidal metal sulfide particles; colloidal lead selenide particles; colloidal cadmium selenide particles; colloidal metal phosphate particles; colloidal metal ferrite particles; any of the above colloidal particles coated with an organic or inorganic layer; protein or peptide molecules; liposomes; or organic polymer latex particles, such as polystyrene latex beads. Preferred particles may be colloidal gold particles.

[0179] Similarly, it should be understood that, in describing exemplary embodiments of the invention, various features of the invention may be combined in a single embodiment, drawing or description thereof in order to simplify the disclosure and aid in understanding one or more of the various aspects of the invention.

[0180] This application also provides various aspects and embodiments in the following statements. In these statements, the phrase “a binder according to statement 1, wherein…” or “a binder according to any one of statements 2 to 6, wherein…” is also disclosed in the simple phrase “in some embodiments…”, and may be replaced therein.

[0181] Statement 1. A binding agent capable of specifically binding to and inhibiting neutrophil elastase (NE). The binding agent comprises an immunoglobulin single variable domain (ISVD), and the binding agent competes with elastase inhibitor 3 (EI3) for binding to NE.

[0182] Statement 2. The binding agent according to Statement 1, wherein the binding agent comprises an immunoglobulin single variable domain (ISVD), said ISVD binding to at least one of NE residues R36, A60, N61, P96, V97, S195, G218, and G219; preferably binding to two or more of residues R36, A60, N61, P96, V97, S195, G218, and G219; more preferably binding to three or more of residues R36, A60, N61, P96, V97, S195, G218, and G219; even more preferably binding to residues R36, A60, N61, P96, V97, S195, G218, and G219. Four or more of S195, G218 and G219; more preferably five or more of residues R36, A60, N61, P96, V97, S195, G218 and G219; more preferably six or more of residues R36, A60, N61, P96, V97, S195, G218 and G219; more preferably seven or more of residues R36, A60, N61, P96, V97, S195, G218 and G219; and even more preferably all of residues R36, A60, N61, P96, V97, S195, G218 and G219.

[0183] Statement 3. A binding agent capable of specifically binding to and inhibiting neutrophil elastase (NE), wherein the binding agent comprises an immunoglobulin single variable domain (ISVD), the ISVD comprising a complementarity-determining region 1 (CDR1) having the sequence shown in SEQ ID NO: 1 (GRTISLYR), a CDR2 having the sequence shown in SEQ ID NO: 2 (INWSGDMT), and a CDR3 having the sequence shown in SEQ ID NO: 3 (TADPKLLPLADSSYGY).

[0184] Statement 4. A binding agent capable of specifically binding to and inhibiting neutrophil elastase (NE), wherein the binding agent comprises an ISVD, the ISVD comprising CDR1, CDR2 and CDR3, each CDR being present in SEQ ID NO: 4, wherein CDR1, CDR2 and CDR3 are annotated according to any one of the numbering systems IMGT, Kabat, Chlotia, Martin or AHo.

[0185] Statement 5. The binder described in Statements 1 to 4 contains or is composed of VHH.

[0186] Statement 6. The binding agent according to any one of claims 1 to 5, wherein the ISVD comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 (QVQLQESGGGLVQAGGSLRLSCVVPGRTISLYRMGWFRQAPGKEREFVAGINWSGDMTDYVDSVKGRFTISRDNAKNTVYLEMNSLKPEDTAIYYCTADPKLLPLADSSYGYWGQGTQVTVSS).

[0187] Statement 7. The binding agent according to any one of Statements 1 to 6, wherein the ISVD comprises the amino acid sequence shown in SEQ ID NO: 4(QVQLQESGGGLVQAGGSLRLSCVVPGRTISLYRMGWFRQAPGKEREFVAGINWSGDMTDYVDSVKGRFTISRDNAKNTVYLEMNSLKPEDTAIYYCTADPKLLPLADSSYGYWGQGTQVTVSS).

[0188] Statement 8. The binder according to any one of Statements 1 to 7, wherein the binder binds to at least a portion of the active site of the NE.

[0189] Statement 9. A nucleic acid molecule comprising a polynucleotide sequence encoding a binding agent according to any one of Statements 1 to 8, or a vector comprising said nucleic acid molecule.

[0190] Statement 10. A cell or virus comprising a nucleic acid molecule or vector as described in Statement 9, optionally wherein the cell is capable of expressing or expressing a binding agent, or the virus is configured to induce the expression of the binding agent in recipient cells infected by the virus.

[0191] Statement 11. A pharmaceutical composition comprising a binder according to any one of Statements 1 to 8, a nucleic acid molecule or carrier according to Statement 9, or a cell or virus according to Statement 10, and a pharmaceutically acceptable carrier, diluent, and / or excipient, wherein the pharmaceutical composition comprises other NE inhibitors.

[0192] Statement 12. The pharmaceutical composition according to Statement 11, wherein the pharmaceutical composition is suitable for administration to a subject via the nasal cavity or lungs, optionally wherein the pharmaceutical composition is formulated as an inhalable dry powder, and optionally wherein the pharmaceutical composition is configured for use in a nebulizer or metered-dose inhaler.

[0193] Statement 13. An apparatus comprising a pharmaceutical composition according to Statement 11 or 12, wherein the apparatus is a nebulizer, a metered-dose inhaler, or a dry powder inhaler.

[0194] Statement 14. A kit, such as a diagnostic kit, comprising a binder according to any one of Statements 1 to 8, a nucleic acid molecule or vector according to Statement 9, or a cell or virus according to Statement 10.

[0195] Statement 15. The binder according to any one of Statements 1 to 8, the nucleic acid molecule or carrier according to Statement 9, the cell or virus according to Statement 10, or the pharmaceutical composition according to Statement 11, for medicinal use.

[0196] Statement 16. Use of the binder according to any one of Statements 1 to 8, the nucleic acid molecule or carrier according to claim 9, the cell or virus according to claim 10, or the pharmaceutical composition according to claim 11 for the prevention or treatment of inflammatory diseases.

[0197] Statement 17. Use of the binder, nucleic acid molecule, carrier, cell or virus, or pharmaceutical composition as described in Statement 16, wherein the inflammatory disease is a lung disease, preferably selected from diseases of mucoid obstructive pulmonary disease, such as cystic fibrosis, chronic obstructive pulmonary disease (COPD), bronchiectasis, ciliary dyskinesia and acute respiratory distress syndrome, inflammatory nasal and sinus diseases, or the inflammatory disease is inflammatory bowel disease, or the inflammatory disease is an inflammatory skin disease.

[0198] Statement 18. Use of the binder according to any one of Statements 1 to 8, the nucleic acid molecule or vector according to Statement 9, the cell or virus according to Statement 10, or the kit according to Statement 14 for the diagnosis of inflammatory diseases in subjects.

[0199] Statement 19. A method for determining the level of active neutrophil elastase in a sample, the method comprising contacting the sample with a binder according to any one of Statements 1 to 8, and detecting at least the neutrophil elastase bound to the binder.

[0200] Statement 20. A method for diagnosing, prognosing, and / or monitoring inflammatory diseases in subjects, optionally any inflammatory disease as defined in Statement 17, said method comprising: - The biological sample obtained from the subject will be contacted with the binder according to any one of statements 1 to 8, and - The level of active neutrophil elastase in a sample is determined by detecting at least the neutrophil elastase bound to the binder.

[0201] Although the invention has been described in conjunction with specific embodiments thereof, many alternatives, modifications, and variations will be apparent to those skilled in the art from the foregoing description. Therefore, the invention is intended to cover the spirit and scope of the appended claims, as well as all such alternatives, modifications, and variations.

[0202] The various aspects and embodiments of the invention disclosed herein may be further supported by the following non-limiting examples.

[0203] Example

[0204] Example 1 - Materials and Methods

[0205] 1. Selection and in vitro characterization of NbE201.

[0206] 1.1. Animal immunization and construction of immune libraries.

[0207] For alpacas ( Lama glamaAnimals were immunized by subcutaneous injection (sc) of 100 µg human sputum elastase (Elastin Products, United States) and Gerbu adjuvant six times, one week apart. Four days after the last immunization, approximately 80 ml of blood was collected from the jugular vein and mixed with ethylenediaminetetraacetic acid (EDTA) to prevent clotting. The blood sample was centrifuged using Lymphoprep centrifuge tubes (Nycomed, Switzerland) to isolate peripheral blood lymphocytes (PBLs). The immune library was constructed according to the protocols described by Pardon et al. (A general protocol for the generation of Nanobodies for structural biology. Nat. Protoc. (2014) doi: 10.1038 / nprot.2014.039) and Vincke et al. (Generation of single domain antibody fragments derived from camelids and generation of manifold constructs. Methods MolBiol. 2012;907:145-76. Doi: 10.1007 / 978-1-61779-974-7_8). Briefly, total RNA was isolated from the PBL using the RNeasy Plus Mini kit (Qiagen, Germany). Then, cDNA was synthesized using 29 µg of total RNA as a template with Oligo(dT)12-18 primers (Waltham, Massachusetts, USA). Next, cDNA was amplified using primers CALL001 (5′-GTC CTG GCT GCT CTT CTA CAA GG-3′ (SEQ ID NO: 5)) and CALL002 (5′-GGT ACG TGC TGT TGA ACT GTT CC-3′ (SEQ ID NO: 6)). These primers amplified the heavy chain antibody gene fragment from the variable region to the CH2 region. Amplification of this region produced two bands: one 900 base pairs (bp) corresponding to the conventional antibody; and the other 700 bp corresponding to the variable region of the heavy chain (VHH, nanobody, or single-domain antibody (sdAb)). The heavy chain variable region was excised from the gel, and DNA was extracted using NucleoSpin Gel and a PCR Clean-up Mini-Prep Kit (Macherey-Nagel, Germany).After amplification using primers PMCF (5'-CTA GTG CGG CCG CTGA GGA GACGGT GAC CTG GGT-3' (SEQ ID NO: 7)) and A6E (5'GAT GTG CAG CTG CAG GAG TCT GGR GGAGG-3' (SEQ ID NO: 8)), the ISVD gene was digested with restriction endonucleases BstEII and PstI and cloned into the pMES3 phage vector, which contains the pelB signal coding sequence for targeting ISVD to the periplasmic space of E. coli.

[0208] Escherichia coli TG1 electrotransformed competent cells (Lucigen, United States) were transformed using a phage plasmid library. The resulting transformants were collected in Luria-Berthani (LB) medium supplemented with ampicillin (100 µg / ml) and 10% (v / v) glycerol and stored at -80°C. This constituted the bacterial library. The percentage of clones in each library containing phage plasmids encoding ISVD was calculated by performing colony polymerase chain reaction (PCR) on 50 randomly selected transformants using specific primers MP57 (5'- TTA TGC TTC CGG CTC GTA TG-3' (SEQ ID NO: 9)) and GIII (5'- CCA CAG ACA GCC CTC ATA G-3' (SEQ ID NO: 10)).

[0209] 1.2. Selection of hNE-specific ISVDs by displaying directional sandwich phages in solution.

[0210] 1.2.1. Phage amplification Take 1 ml of aliquots of the bacterial library and grow them in 2XTY medium supplemented with ampicillin (100 µg / ml) and glucose (0.1%, %w / v, final concentration) until the optical density (OD) is reached. 600nm The concentration was reached to 0.8. Then, as previously described (Pardon et al., Nat. Protoc. 2014, doi: 10.1038 / nprot.2014.039), cells were infected with M13K07 phage (ThermoFisher Scienctific, United States) at a 20-fold multiplicity of infection. Phage virus particles were recovered by polyethylene glycol-6000 precipitation and resuspended in sterile phosphate-buffered saline (PBS) at pH 7.4. This constructed a phage library. The phage concentration was estimated by measuring absorbance at 260 nm.

[0211] 1.2.2. Directional sandwich phage display and selection in solution A solution-based directional sandwich panning technique was designed to select ISVDs that specifically target the active site of hNE. ISVD NbE34 was captured by biotinylation, and three rounds of panning were performed on hNE immobilized on the surface of streptavidin-coated magnetic Dynabeads M280 (ThermoFisher Scientific, United States). NbE34 is an hNE-specific ISVD that does not bind to the enzyme's active site. NbE34 was selected from a set of non-inhibitory ISVDs based on their ability to expose the active site of hNE to a phage-exposed ISVD library for a sufficient time for the panning process, thereby allowing for library enrichment with inhibitory clones. NbE34 has the sequence shown in SEQ ID NO: 15 (QVQLQESGGGLVQPGGSLRLSCAASGFTLDYHAIGWFRQAPGKEREGVSCISSSGGRADYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATRCAVVGGTYYYGMDYWGKGTQVTVSS).

[0212] The following NbE34 sequence was used: QVQLQESGGGLVQPGGSLRLSCAASGFTLDYHAIGWFRQAPGKEREGVSCISSSGGRADYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATRCAVVGGTYYYGMDYWGKGTQVTVSSAAAYPYDVPDYGS (SEQ ID NO: 21) (this sequence contains the NbE34 sequence of SEQ ID NO: 15 and the HA tag). Therefore, this paper discloses a method for in vitro panning of hNE immobilized on beads using NbE34. After each step, beads were recovered for two minutes using 1.5 ml Eppendorf tubes on a magnetic rack. All incubation steps were performed at room temperature on a vertically rotating shaker. Washing was performed using 500 µl of phosphate-buffered saline solution and 0.01% detergent solution (PBST, containing 0.01% (v / v) Tween 20 PBS). First, 0.04 mg of beads were functionalized with 4 µg of chemi-biotinylated NbE34 in 1 ml PBS. After washing five times, the beads were incubated with 5 µg of hNE in 500 µL PBS. Negative control tubes, also functionalized with NbE34 but without hNE, were also prepared. Then, the beads in each tube were incubated in 500 µL blocking buffer. In rounds 1, 2, and 3, the blocking buffer consisted of 0.1% (w / v) casein, 0.1% (w / v) bovine serum albumin, or protein-free blocking buffer (ThermoFisher Scientific, United States). Next, 2x10 [unclear text - likely a continuation of the previous sentence] from the library... 11 One phage was incubated for 1 hour in positive and negative test tubes. Beads were washed at least 10 times with PBST (PBS containing 0.05% (v / v) Tween 20), then eluted with 200 μL triethanolamine (TEA) pH 11 for 10 min and neutralized with 200 μL triaminomethane hydrochloride (Tris-HCl) 1M pH 8. The resulting phage subcell was re-expanded in 15 ml of TG1 cells in the exponential growth phase, grown in LB medium supplemented with ampicillin (100 µg / ml) and glucose (0.2%, %w / v). The cells were then dipped in 10 µl of phage solution (containing 10% (v / v) Tween 20)... 7 Infected with phages, incubated for 1 hour without stirring, then centrifuged at 3000 x g for 15 minutes. The precipitate was resuspended in fresh 2xTY medium supplemented with ampicillin (100 µg / ml) and kanamycin (70 µg / ml), and incubated at 37°C. oIncubate overnight at C20 with orbital oscillation (250 rpm). Perform phage precipitation as described above and use it for the next round of panning. During each round of panning, take 10 µl of eluted phage sample to infect 100 µl of TG1 cells, then serially dilute the TG1 cells until reaching 10⁻⁶. 7 Diluted 1-2 times and inoculated onto LB agar supplemented with ampicillin (100 µg / ml) and glucose (0.2%, %w / v). The enrichment of phages exposed to hNE-specific ISVDs was estimated by comparing the number of colonies at a given dilution in positive tubes (containing hNE in the panning step) and negative tubes (i.e., not containing hNE in the panning step).

[0213] 1.3. Screening using enzyme-linked immunosorbent assay (ELISA).

[0214] Ninety colonies from each round of panning were randomly selected using an EasyPick Microlab STARlet Hamilton workstation (Lincoln, United States) on the Robotein platform at the Protein Engineering Center of Liège University (http: / / www.robotein.ulg.ac.be). Each colony was inoculated into 100 µl of LB medium supplemented with ampicillin (100 µg / ml), glucose (0.2%, %w / v), and glycerol (10%, %v / v) in 96-well round-bottom plates (Greiner Bio-One, Belgium). Six wells were filled with medium only as a negative control. The plates were incubated at 37°C. o Incubate overnight at 37°C with shaking (220 rpm). The next day, inoculate each well with 20 µl of 1 mL of 2xTY medium containing ampicillin (100 µg / mL) and glucose (1%, %w / v) into a 96-well deep-well plate and incubate at 37°C. o Incubate at C with orbital oscillation (220 rpm) for 3 hours. Next, induce ISVD production by adding isopropyl β-D-1-thiogalactoside (IPTG) to a final concentration of 1 mM, and plate the mixture at 37°C. o Incubate at C with orbital oscillation (220 rpm) for 3 hours. Then, centrifuge the plates at 3000g for 20 minutes to obtain cytoplasmic extract (CE) by freeze-thaw cycling the precipitate at -80°C. Resuspend the CE in 100 µl of sterile PBS and centrifuge briefly. The specificity of CE for hNE for each clone was determined by CE-ELISA. Briefly, 100 µl of hNE (1 µg / ml) was incubated at 4°C with orbital oscillation (220 rpm) for 3 hours. oAdsorption was performed overnight at C, and the plates were immobilized on multisorp 96-well plates (ThermoFisher Scientific, United States). Each clone was used with PBS as a blank control. After washing five times with 0.001% (%v / v) PBST, the wells were blocked for 1 hour at room temperature (RT) with 200 µl of 1% (%w / v) PBS containing skim milk blocking buffer, followed by incubation for 1 hour at room temperature with 100 µl of 5-fold diluted CE (in PBS). The plates were then washed 10 times and stained with mouse anti-His antibody (BioLegend, United States) and goat anti-mouse antibody conjugated with alkaline phosphatase (BioLegend, United States) to develop the bound ISVD. Both antibodies were used at a 1:2000 dilution and incubated for 1 hour at room temperature. After each antibody treatment, the plates were washed 10 times with 0.001% (%v / v) PBST. The colorimetric reaction was performed in alkaline phosphate buffer (100 mM Tris, 50 mM MgCl26H2O, 100 mM NaCl, pH 9.5) containing 2 mg / ml p-nitrophenyl phosphate (pNNP). Absorbance at 405 nm was measured after 5–10–15–20–40 minutes using a Molecular Devices SpectraMax M2e microplate reader. A clone was considered positive when its absorbance was at least twice that of its corresponding blank control. Plasmid DNA was isolated from single positive clones and sequenced using specific primers MP57 (5'-TTA TGC TTC CGG CTC GTA TG-3' (SEQ ID NO: 9)). ISVDs with highly similar complementarity-determining region 3 (CDR3) sequences were grouped into families. One ISVD from each family was selected for production and characterization (i.e., the ability to suppress hNE).

[0215] 1.4. Produce and purify one ISVD for each family.

[0216] Phage particles encoding the target ISVD were transformed into chemically competent *E. coli* WK6 cells. The transformed cells were plated onto LB agar containing ampicillin (100 mg / L). One colony was picked and diluted in 100 mL of ampicillin (100 mg / L). -1 Pre-culture was performed overnight (ON) at 37°C in LB medium. The pre-culture (5 mL) was then used to inoculate 500 mL of Terrific Broth medium supplemented with glucose (0.1% v / v) and ampicillin (100 mg / L). Bacterial growth was carried out at 37°C until 600 cells / mL were reached. nmabsorbance at (A) 600nm Between 2 and 3. Then add 1 mM IPTG (final concentration) and continue oscillating culture at 28°C. Harvest cells by centrifugation (6000 g for 5 min) and prepare periplasmic extract by osmotic shock as previously described (Skerra, A. & Plückthun, Science, 1988, doi: 10.1126 / science.3285470). The periplasmic extract was then filtered through a 0.45 µm cutoff membrane and loaded onto 5 mL of immobilized metal affinity chromatography (IMAC) NiPDC resin (Affilland, Liège, Belgium). Wash the resin with 0.5 M pH 8 potassium phosphate buffer (KP buffer) containing 50 mM imidazole, and then elute Nb with 0.5 M pH 8 KP buffer containing 375 mM imidazole. Imidazole was removed using a G25 Sephadex column (Sigma Aldrich) and 0.5 M sodium buffer (NaP buffer) at pH 7 as the run buffer. The purity and integrity of the ISVD were verified by Coomassie Brilliant Blue stained SDS-PAGE, mass spectrometry (ESI-Q-TOF), and SEC-MALS. The theoretical extinction coefficient was calculated based on the amino acid sequence using Expasy prot parameters, and the concentration was determined by UV absorbance at 280 nm using this theoretical extinction coefficient.

[0217] 1.5. Mass production of NbE201, NbE201-C terminal and Nb-H7S-Nter-P1.

[0218] The NbE201 gene was cloned into the pHEN6 vector using PstI and BstEII restriction endonuclease sites. The resulting vector was used to transform chemically competent *E. coli* WK6. The C-terminus of NbE201 is NbE201 fused to the following sequence (containing the linker of SEQ ID NO: 16 and a C-terminal cysteine ​​residue for conjugation with PEG-maleimide): SPSPTPPTPSPSTPPC (SEQ ID NO: 22). Nb-H7S-Nter-P1 is a chimeric Nb containing a peptide P1 fused to the N-terminus of Nb-H7S, which is specific for the β-lactamase BlaP (i.e., Nb-H7S does not bind to NE). P1 consists of an inhibitory peptide sequence (β-hairpin) and a β-stem derived from bovine Ab BLV1H12 (Liu, T. et al., doi.org / 10.1021 / ja5130786). The sequence of P1 is: TSVHQETKK MC TASIPPQC Y YNYEWHVDV (SEQ ID NO: 11), where C -C The disulfide bond is indicated; the sequence of the inhibitory peptide, MCTASIPPQCY (SEQ ID NO: 12), is underlined. NbE201-Cter and Nb-H7S-Nter-P1 were produced and purified using the same protocol as described above in 3 L Terrific Broth (TB) medium.

[0219] 1.6. Inhibition of hNE activity

[0220] 1.6.1. Inhibiting the hydrolysis of chromogenic substrates To determine the inhibitory activity of NbE201, the residual activity of hNE in the presence of Nb was measured at different Nb:hNE ratios. hNE was incubated with the substrate N-succinyl-Ala-Ala-Ala-p-nitroaniline and different concentrations of ISVD. Briefly, hNE (Elastin Products Company, EPC, United States) was incubated at 1 mg / mL... -1 Resuspend in 50 mM sodium acetate buffer (pH 5) containing 100 mM NaCl and store at 4°C. Dissolve the substrate succinyl-Ala-Ala-Ala-p-nitroaniline at 1 mM in 0.1 M Tris (pH 7.5) containing 0.5 M NaCl and 0.01% (%w / v) NaN3 and store at 4°C. Mix 230 µl of substrate solution (1 mM) with 10 µl of 9 µM bovine serum albumin (BSA, Sigma-Aldrich, Germany) and incubate in a clear-bottomed 96-well plate (Greiner Bio-One, Belgium) at 25°C for 20 min. Then, add ISVD at final concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, and 200 nM. hNE was then immediately added to the mixture to a final concentration of 50 nM. Absorbance at 410 nm was measured every minute for 60 minutes using a SpectraMax M2e microplate reader (Molecular Devices, California, USA). Initial rates were compared with those without ISVD in the presence of different Nb:hNE ratios to determine residual activity. The delay between hNE addition and the first measurement was approximately 100 seconds. All experiments were performed triplicate using three independent batches of NbE201. In some experiments, ISVD was pre-incubated with hNE for 15 minutes before the complex was added to the substrate.

[0221] The inhibitory activity of three other hNE inhibitors was determined using the same setup for comparison with NbE201: α-1-antitrypsin (AAT, Sigma-Aldrich, Germany), elastase inhibitor 3 (EI3, or MeOSuc-Ala-Ala-Pro-Val-CMK, Calbiochem, Sigma-Aldrich, Germany), and cevelextastat (R&D Systems, UK). Briefly, the substrate used was succinyl-Ala-Ala-Ala-p-nitroaniline (1 mM), dissolved in 0.1 M Tris pH 7.5 containing 0.5 M NaCl and 0.01% (%w / v) NaN3. The hNE concentration used was 50 nM, and different inhibitor concentrations were used to obtain different inhibitor:hNE molar ratios. Absorbance was measured at 410 nm every minute for 60 minutes. The initial rate of activity was determined by comparing the initial rate of activity with different inhibitor-to-hNE ratios with that without inhibitor. The time between the addition of hNE and the first measurement was approximately 100 seconds. The activity of some hNEs in the presence and absence of NbE201 was also measured in quartz microcuvettes. The substrate succinyl-Ala-Ala-Ala-p-nitroaniline 1 mM was incubated at 25°C for 20 minutes. Then, two different concentrations of NbE201 (C...) were added. f = 40 nM and 75 nM) and hNE (Cf = 50 nM) were mixed. hNE activity was monitored at 410 nm for 10 min using a Specord 50 Plus spectrophotometer (AnalytikJena), and data were collected using WinAspectPlus software. In BSA (C f Measurements were performed in the presence of hNE (=346 nM). The time between the addition of hNE and the first measurement was approximately 14 seconds.

[0222] The Km of hNE for succinyl-Ala-Ala-Ala-p-nitroaniline was determined by incubating 50 nM hNE with different substrate concentrations (12, 24, 48, 96, 192, 391, 586, 781, 1562, 2344, and 3125 mM) in the presence of 346 nM BSA. The reaction was monitored as described above. The initial hydrolysis rate (v0) was plotted as a function of [S] and linearized using the Hanes-Woolf method to obtain the Km value.

[0223] The Km of mNE for MeOSuc-Ala-Ala-Pro-Val-AMC was determined by incubating 14.7 nM mNE with different substrate concentrations (0.025, 0.5, 0.1, 0.2, 0.4, 0.6, 0.8, 1.2 mM). The reaction was monitored as described above. The initial hydrolysis rate (v0) was plotted as a function of [S] and linearized using the Hanes-Woolf method to obtain K. m value.

[0224] 1.6.2. Inhibit the hydrolysis of elastin (the natural substrate of hNE). The residual activity of hNE was measured in the presence of elastin-Congo red (Sigma-Aldrich, Germany). hNE (132 nM) was mixed with an elastin-Congo red solution (10 mg / L). mL -1 The samples were dissolved in 0.1 M Tris (pH 7.5) containing 0.5 M NaCl and 0.01% (w / v) NaN3, and incubated with different concentrations of NbE2O1 or other inhibitors (e.g., AAT, EI3, and cevelexat). The samples were incubated at 37°C with orbital oscillation (900 rpm) for 16 h. The samples were then centrifuged at 90,000 g for 30 min at 4°C to separate insoluble elastin from the digested soluble molecular fragments. The soluble fractions were transferred to 384-well clear plates, and the absorbance of Congo red at 495 nm was measured using a SpectraMax M2e microplate reader (Molecular Devices, California, USA). The absorbance of samples with different inhibitor:hNE molar ratios was compared with that of samples without inhibitors to determine residual hNE activity. Elastin-Congo red samples without elastase were used as blank controls.

[0225] 1.7. Specificity of NbE201 to hNE.

[0226] To determine whether NbE201 is specific for hNE, we first investigated its ability to inhibit several serine proteases: porcine pancreatic elastase (pPE, sequence identity 39.2%, from Sigma), mouse neutrophil elastase (mNE, sequence identity 74.9%, from EPC), and two other human serine proteases present in the lungs: protease 3 (hPR3, from EPC, sequence identity 54.7%) and cathepsin G (hCG, from Calbiochem, sequence identity 36.7%). The substrates used were: succinyl-Ala-Ala-Ala-p-nitroaniline (1 mM, dissolved in 0.1 M Tris containing 0.5 M NaCl and 0.01% (w / v) NaN3 at pH 7.5, purchased from Sigma-Aldrich), for pPE; MeOSuc-Ala-Ala-Pro-Val-AMC (100 µM, dissolved in 50 mM Tris containing 1 M NaCl and 0.05% (w / v) Brij-35 at pH 7.5), for mNE; N-succinyl-Ala-Ala-Pro-Phe p-nitroaniline (0.45 mM, dissolved in 100 mM HEPES at pH 7.5, purchased from Sigma-Aldrich), for hCG; and Boc-Ala-Ala-Nva-SBzl (0.22 mM, dissolved in 0.5 M NaCl and 100 µM Tris containing 0.1 M NaCl and 0.01% (w / v) NaN3 at pH 7.5), for mNE; and Boc-Ala-Ala-Nva-SBzl (0.22 mM, dissolved in 0.5 M NaCl and 100 µM Tris containing 0.01% (w / v) NaN3 at pH 7.5), for mNE; and Boc-Ala-Ala-Nva-SBzl (0.22 mM, dissolved in 0.1 M Tris containing 0.5 M NaCl and ... DTNB and DMSO (10% in 100 mM MOPS, pH 5.0, purchased from EPC) were used for PR3. mNE (rmELA2, purchased from R&D Systems, UK) was first activated according to the supplier's information. Briefly, mNE was incubated with recombinant mouse active cathepsin C / DPPIy (rmCathepsin C, purchased from R&D Systems) at 37°C for 2 h in 50 mM MES, 50 mM NaCl, pH 5.5. The enzyme concentrations of PPE, mNE, hCG, and hPR3 in the assay were 6 nM, 14.7 nM, 25 nM, and 5 nM, respectively. Different NbE201:enzyme molar ratios were tested. Substrate hydrolysis and residual activity were determined as explained in the hNE inhibition assay above. Cross-reactivity was also investigated using biolayer interference (BLI).

[0227] 1.8. The affinity of NbE201 for hNE and mNE was determined by BLI experiment.

[0228] The affinity of NbE201 for hNE and mNE was measured via BLI using an Octet HTX instrument with various settings. NbE201 was generated in a pHEN25 carrier and site-specifically biotinylated at its C-terminal Cys terminus using EZ-Link maleimide-PEG2-biotin (ThermoFisher) and tris(2-carboxyethyl)phosphine, immobilized on agarose CL-4B (Sigma-Aldrich), and incubated with shaking in 50 mM NaPi buffer (pH 7) and 2 mM EDTA at room temperature for 2 hours and 30 minutes. For hNE, biotinylated NbE201 (0.5 µg) was added according to the supplier's instructions. mL -1 The hNE solution was immobilized on a streptavidin biosensor (SA, Sartorius). The biosensor was then immersed in hNE solutions of different concentrations (25 – 6.25 – 3.125 – 1.5163 – 0.753 nM). Binding and dissociation kinetics were measured at 180 sec and 300 sec in 10X KB buffer. All experiments were performed at 30°C in 96-well or 384-well black-bottomed polypropylene microplates (Greiner Bio-One, Belgium) with orbital oscillation (1500 rpm). The obtained sensor maps were fitted to a 1:1 model to derive the binding rate constant (kΩ). on ) and dissociation rate constant (k off and equilibrium dissociation constant (K) D =k off / k on Two independent experiments and one independent experiment repeated twice were conducted. K D The error is calculated using the following formula:

[0229] Where σ is the error, K D k is the dissociation constant at equilibrium. off k is the dissociation constant. on is the binding constant.

[0230] For mNE, NbE201_Cter_Biot was immobilized on the SA biosensor (Sartorius) via C-terminal biotin, as instructed by the supplier. The biosensor was then immersed in mNE solutions of varying concentrations (100 – 50 – 25 – 12.5 – 6.25 – 3.125 nM). Binding and dissociation kinetics were measured at 300 sec and 600 sec, respectively. All experiments (for hNE and mNE) were performed at 30°C in 384-well black-bottomed polypropylene microplates (Greiner Bio-One, Belgium) with orbital oscillation (1000 rpm). The obtained sensor maps were fitted to a 1:1 model using Octet software version 10.0 (Sartorius) to derive the binding rate constant (k). on ) and dissociation rate constant (k off and equilibrium dissociation constant (K) D =k off / k on ).

[0231] 1.9. Binding of NbE201 to other serine proteases.

[0232] The specificity of NbE201 for hNE was also investigated by BLI. Biotinylated NbE201 (10 µg / mL) was immobilized on a streptavidin biosensor (SA, Sartorius) according to the supplier's instructions. The biosensor was then immersed in a 50 nM solution of serine proteases. The proteases tested were human cathepsin G (hCG), human protease 3 (hPR3), mouse pancreatic elastase (mPE), mouse neutrophil elastase (mNE), and porcine pancreatic elastase (pPE). hNE was used as a positive control. Binding and dissociation were measured at 70 sec and 320 sec in kinetic buffer (KB, Sartorius). Experiments were performed at 30°C in 384-well black-bottomed polypropylene microplates (Greiner Bio-One, Belgium) with orbital oscillation (1000 rpm).

[0233] 1.10. Competitive binding of NbE201 to α1-antitrypsin (AAT), elastase inhibitor III (EI3), and Nb-H7S-Nter-P1. The competitive binding of NbE201 and hNE to (i) AAT, (ii) EI3, and (iii) Nb-H7S-Nter-P1 complexes was also determined using a Octet HTX device via biolayer interferometry (BLI). Biotinylated NbE201 (10 µg / ml, dissolved in KB buffer) was immobilized on a streptavidin biosensor (SA, Sartorius) according to the supplier's instructions. The biosensor was quenched using 1 µg / mL biocytin. The biosensor was then immersed in 250 nM hNE, which was pre-incubated with a saturated concentration of an active site competitor (1.5 µM AAT, 15 µM MEI3, or 1.5 µM Ab-H7S-Nter-P1), or without pre-incubation. Binding was measured for 280 seconds in kinetic buffer (KB buffer, Sartorius). Experiments were performed at 30°C in 384-well black-bottomed polypropylene microplates (Greiner Bio-One, Belgium) with shaking (1000 rpm).

[0234] 1.11. Thermodynamic stability of NbE201

[0235] 1.11.1. Sample Preparation The NbE201 sample was incubated overnight at 25°C in 0.05 M NaP buffer (pH 7) with increasing concentrations of urea (Sigma-Aldrich). The protein concentration was 0.2 mg / mL. -1 The denaturant concentration in each sample was determined by refractive index measurement using an Atago (Tokyo, Japan) R5000 handheld refractometer (Pace, CN, 1986, doi.org / 10.1016 / 0076-6879(86)31045-0). Intrinsic fluorescence and far-ultraviolet circular dichroism (UV-CD) spectra were measured using the same samples. Blank samples (Nb-free) were prepared for each 1 M urea to create reference curves to correct for the contribution of (buffer + urea) to the fluorescence and circular dichroism (CD) signals at each urea concentration.

[0236] 1.11.2. Measurement of fluorescence and far-UV-CD signals / spectroscopy 。 Use a multi-cuvette holder with temperature control and 10 A Cary Eclipse fluorometer (Agilent, Santa Clara, USA) with a 0.4 mm optical path quartz miniature cuvette was used to record intrinsic fluorescence spectra from 305 nm to 440 nm at 25°C after excitation at 280 nm, with excitation and emission slits at 5 nm. The voltage applied to the detector was 710 V. Data was acquired every 0.5 nm at a scan rate of 30 nm·min. -1 .

[0237] Far-UV-CD spectra of native and denatured states (i.e., in 9 M urea) from 190 nm to 260 nm were recorded in a 0.2 mm path length quartz cuvette at 25°C. 606 data points were recorded with a data interval of 0.1 nm, an integration time of 2 s, a bandwidth of 1 nm, and a scan rate of 50 nm / min. The development transition at 207 nm was monitored by far-UV-CD in the 0.2 mm path length quartz cuvette. At each denaturant concentration, 37 data points were acquired with a bandwidth of 1 nm, a readout frequency of 5 s, and an integration time of 4 s, and the average value was taken. Measurements were performed using a Jasco J-810 spectrophotometer (JASCO, Lisses, France) equipped with a Peltier element holder.

[0238] 1.11.3. Data Analysis 。 The intrinsic fluorescence spectrum was fitted using a five-parameter Weibull function (from SigmaPlot 5.0 software) to determine the wavelength (λ) corresponding to the highest fluorescence intensity. max The fluorescence intensity change at 337 nm was also used to monitor the unfolding transition. This was based on the two-state (N) model proposed by Dumoulin et al. (doi: 10.1110 / ps.34602). The U) model and Equation 1 were used to analyze the obtained transformation curves: y obs = {(Y N + p [x]) + (Y U + q [x]) exp(-a)} / (1 + exp (-a)) (1), In Formula 1, x is the denaturant concentration, and a = (ΔG) ° NU(H2O) - m [D]) / RT,y obs For the monitoring signal at a given denaturant concentration (i.e., λ)max F 337nm or CD 207nm ), Y N and Y U These are the values ​​of these signals in the native and denatured states, respectively, without a denaturing agent. ΔG ° NU(H2O) is the free energy difference between the folded and unfolded states at 25°C; m is a measure of the dependence of free energy on denaturant concentration, and [D] is the denaturant concentration. p and q are the slopes of the baselines before and after unfolding, respectively; R is the gas constant; and T is the absolute temperature. m The denaturant concentration at the midpoint of the denaturation curve ([U] / [N] = 1) is defined as ΔG. ° NU(H2O) / m.

[0239] Normalize the transformation using formula (2): y = {y obs - (Y N + p [x])} / {(Y U + q [x]) - (Y N + p [x])} (2)

[0240] 1.12. Thermal stability of NbE201.

[0241] 1.12.1. Sample Preparation and Measurement NbE201 samples were prepared in 0.05 M NaP buffer at pH 7, with a protein concentration of 0.2 mg / mL. -1 All experiments were performed in a JASCO J-810 CD spectrophotometer with a 1 mm quartz cell and a Peltier element holder. Mineral oil was added to the top of the samples to prevent evaporation. CD spectra of native and denatured proteins from 190 nm to 260 nm were recorded at 25°C and 97°C. Sixty-one data points were recorded with a data interval of 1 nm, a data separation interval of 0.1 nm, an integration time of 2 s, a bandwidth of 1 nm, and a scan rate of 50 nm / min. Thermally induced expansion at 206 nm was monitored by far-UV-CD. The temperature was controlled at 0.5 °C. min -1 The heating rate was linearly increased from 25°C to 97°C, with data points collected every 0.3°C. The reversibility of this phenomenon was determined by cooling the sample from 97°C to 25°C at the same rate. Data acquisition was performed at a readout frequency of 0.05 s. -1The integration time was 4 s and the bandwidth was 1 nm. The temperature in the cuvette was monitored using a thermocouple (PT200 differential thermometer, IMPOTEelectronics, Albertslund, Denmark).

[0242] 1.12.2. Data Analysis. Based on bistate (N) The U) model and Formula 1 are used to analyze the obtained conversion curve, where x is the temperature during the analysis period, and a = -(ΔH) m (1-x / T m )) / RT (doi:10.1016 / j.jmb.2008.11.046), y obs The CD signal at 206 nm at a given temperature (x), Y N and Y U These are the signal values ​​at 273 K in the natural and modified states, respectively. p and q are the slopes of the baselines before and after the conversion, respectively, R is the gas constant, and T is the absolute temperature. m It refers to the temperature during the conversion, ΔH. m It is T m The enthalpy value at time T. Since the transformation is not completely reversible, only the value known as apparent T is considered. m (T m app Tm of ) is normalized using Equation 2.

[0243] 1.13. Stability of NbE201 to hNE.

[0244] NbE201 (26.4 µM) was incubated alone or in the presence of hNE ([NbE201]:[hNE]=4:1) at 37°C. Aliquots (10 µL) taken at different time intervals were mixed with 4 μL of sample loading buffer (Tris-HCl (pH 6.8), SDS denaturant (4%, %w / v), β-mercaptoethanol (5%, %v / v), bromophenol blue (0.01%, %w / v), glycerol (20%, %v / v), H2O) and stored at -20°C until SDS-PAGE analysis using 4–20% Mini-PROTEAN TGX protein gels, 10 wells, 50 μL (Bio-Rad, Germany). The gels were then stained with Coomassie Brilliant Blue, destained in water, and imaged using a Gel Doc EZ imager (Biorad). The relative content of intact protein was quantified by density analysis using the Gel Doc EZ Imager program, with the band intensity at time zero as a relative standard. hNE was incubated alone under similar conditions, and aliquots were taken at different time points. The activity of hNE was determined using N-succinyl-Ala-Ala-Ala-p-nitroaniline as a substrate, as described above.

[0245] 1.14. Crystal structures of NbE2O1 alone and in complexes with hNE.

[0246] 1.14.1. Crystallization conditions of the complex ： Crystals were grown at 20°C using a hanging drop vapor-phase diffusion method. The droplet contained 0.2 µL of a complex formed from hNE and NbE2O1 at a concentration of 15 mg·mL⁻¹. -1 The crystals were prepared with 0.2 µL of 0.1 M HEPES pH 7 buffer containing 10% polyethylene glycol 5000 monomethyl ether and 5% tacsimate. The crystals were then transferred to a cryoprotectant solution containing 33% (v / v) polyethylene glycol 6000 and 33% (v / v) glycerol and frozen in liquid nitrogen.

[0247] 1.14.2. Conditions for the single crystallization of NbE2O1: Crystals were grown at 20°C using a hanging drop vapor-phase diffusion method. The droplet contained 0.2 µL of a solution with a concentration of 15 mg·mL⁻¹. -1 The solution contained NbE201 and a cryoprotectant solution of 0.2 M calcium acetate containing 0.2 µL PBS buffer (pH 7.4), 10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, and 2.7 mM KCl, as well as 0.1 M sodium methylarsinate (pH 6.5) containing 18% (v / v) polyethylene glycol 8000 and 33% (v / v) glycerol.

[0248] 1.14.3. Data collection, phase determination, model building, and refinement are performed for both structures. Diffraction data were collected using the Proxima 1 beamline at the Soleil synchrotron (Saint Aubin, France). The data were indexed, integrated, and scaled using the XDS package (Kabsch W., 2010, doi.org / 10.1107 / S0907444909047337). Initial phases were obtained using molecular substitution with the structure of hNE alone (PDB 3Q76) and / or the ISVD-specific structure of the mouse Vsig4 protein (PDB 5IMO) via the Phaser crystallography program (McCoy AJ et al., 2007, doi.org / 10.1107 / S0021889807021206). The structure was constructed using the Coot program (Crystallographic Object-Oriented Toolkit) (Emsley P. et al., 2010, doi.org / 10.1107 / S0907444910007493) and refined using BUSTER-TNT (Blanc E. et al., 2004, doi: 10.1107 / S0907444904016427). The figures were drawn using PyMOL (PyMOL Molecular Graphics System, Mac OS X Enhanced Edition 2.4.1, Schrödinger, LLC.).

[0249] 2. PEGylation of NbE201, stability under different stresses, inhibition of hNE in sputum of CF and COPD patients, and pharmacokinetics.

[0250] 2.1. PEGylation and purification of NbE201

[0251] A 0.063 mM NbE201-Cter solution was exposed to 1 mM tris(2-carboxyethyl)phosphine (TCEP) (Merck, Kenilworth, USA) linked to agarose beads and 0.252 mM linear 10 or 20 kDa polyethylene glycol (PEG)-maleimide or branched 40 kDa PEG-maleimide (Nof Corporation, Tokyo, Japan). The reaction was carried out at 150 rpm on a stirring plate at room temperature in 2 mM EDTA (Merck), 50 mM sodium phosphate buffer, pH 7, for 2 hours and 30 minutes. The solution was then centrifuged at 1000 rpm for 1 minute to precipitate the agarose beads, and the supernatant was collected. The PEGylated product was purified by rapid protein liquid chromatography (FPLC) (Akta purifier 10, Cytiva, Chicago, USA) using a cation exchange column (ResourceS, 1 ml, Cytiva). The primary buffer was 20 mM succinic acid, 5 mM NaCl solution, pH 4.6; the elution buffer was 20 mM succinic acid, 350 mM NaCl solution, pH 4.6. The solution was dialyzed against the primary buffer (3 kDa MWCO membrane, Carl Roth, Karlsruhe, Germany) and filtered through a 0.2 µm polyvinylidene fluoride (PVDF) membrane (Carl Roth). The solution was injected into the column and eluted with 20 ml of gradient elution buffer from 0% to 100% (3 ml / min). The purified PEGylated product was collected using a Frac-920 system (Cytiva). The sample was then transferred to its initial sodium phosphate buffer (50 mM, pH 7) and concentrated to the desired concentration using an ultrafiltration tube (Vivaspin Turbo 3 kDa, Sartorius).

[0252] 2.2 hNE enzyme activity assessment and inhibition.

[0253] 2.2.1 Evaluation of the inhibition of proteolytic activity of purified hNE. By tracking color development hNE Substrate N-succinyl-alanine-alanine-proline-valine-p-nitroaniline (suc- Ala - AlahNE activity was assessed by evaluating the degradation of Pro-Val-pNA (elastin product, Owensville, Missouri, USA). Absorbance was measured at 410 nm using a spectrophotometer (Spectramax ID5, Molecular Devices, San Jose, USA). The substrate was diluted to 1.5 mM in Tris buffer (0.1 M Tris, 0.5 M NaCl, 0.01% NaN3, pH 7.5), and 200 µL of this solution was added to the wells of a 96-well plate and heated at 37°C. The purified hNE (elastin product) was reconstituted to a concentration of 0.1 mg / mL on ice in 0.05 M sodium acetate (NaOAc), 0.1 M NaCl buffer, pH 5. 10 µL of this hNE solution was added to each well, and substrate degradation was monitored over 20 minutes to verify hNE activity in the wells. Then, ISVD was added to the wells at the desired hNE:ISVD ratio, and substrate degradation was tracked over 50 minutes. Regression lines were plotted based on absorbance measurements, the slope was calculated, and expressed as the percentage of hNE activity without ISVD.

[0254] 2.2.2 Assessment of the inhibition of proteolytic activity in sputum of patients with CF, COPD, and ciliary dyskinesia 。 Sputum was collected from patients aged 18 years and older with cystic fibrosis, COPD, or ciliary dyskinesia during monthly physiotherapy consultations, without special induction. The sputum collection procedure was evaluated by the local ethics committee (comité d'Ethique Hospitalo-Facultairedes Cliniques universitaires Saint-Luc) and approved under Belgian registration number B4032021000071.

[0255] Because hNE induces various lung pathologies characterized by chronic inflammation, other conditions such as COPD, AAT deficiency, severe asthma, bronchiectasis, and infiltrative lung disease may benefit from NbE201 treatment. Therefore, the inhibitory capacity of NbE201 (-PEG10 / 40) was evaluated in sputum samples from patients with CF, ciliary dyskinesia, and COPD.

[0256] The suc- at 410 nm was tracked using a spectrophotometer. Ala - Ala -Pro-Val-pNA degradation (color development) hNEThe absorbance of the substrate was used to assess the hydrolytic activity of sputum proteins. Sputum was classified as mucous, mucopurulent, or purulent (Serisier et al., 2009, Respir Res, 10(1): 63. Doi: 10.1186 / 1465-9921-10-6). According to the experiment, 10 to 20 mg of sputum was weighed and placed in a 96-well plate. ISVD was then added to the sputum sample and pre-cultured at 37°C for different time periods. The hNE substrate was diluted to a concentration of 1 mM in Tris buffer (0.1 M Tris, 0.5 M NaCl, 0.01% sodium azide (NaN3), pH 7.5), and 50 µl of the solution was added to the well. Substrate degradation was tracked over 50 minutes. To estimate the elastase activity in each sputum sample, different amounts of sputum ranging from 1 to 5 mg were weighed and placed in 96-well plates. The activity was compared to that of the hNE standard curve, where 2 to 20 µl increments of hNE solution were added to the substrate in Nb-free wells, as described in section 2.2.1. The slope of the regression line was then calculated based on absorbance measurements and expressed as a percentage of ISVD-free sputum protein hydrolysis activity.

[0257] These inhibition experiments will provide the IC50 values ​​of NbE201 after exposure to sputum at different time points (0, 1, 4, and 24 hours). 50 The values ​​were compared with the IC50 values ​​after exposure to NbE201-PEG10, NbE201-PEG40 and AAT (Bio-Techne, Minneapolis, USA). 50 The values ​​are compared.

[0258] 2.3 Stability of NbE201 and its PEGylated counterparts under physical stress.

[0259] Prepare 0.25 mg / ml solutions of NbE201, NbE201-PEG10 or NbE201-PEG40 and dispense them for use under the following conditions: control, stirring with a magnetic rod for 1 or 4 hours, nebulization, and 5 or 10 freeze-thaw cycles.

[0260] 2.3.1 Stability of stirring. Place 100 µl of 0.25 mg / ml ISVD solution into a 1 ml glass vial and stir at 200 rpm for 1 hour or 4 hours using a 2x5 mm magnetic rod (VWR, Radnor, USA).

[0261] 2.3.2 Stability of atomization. 300 µl of 0.25 mg / ml ISVD solution was nebulized using a MeSH nebulizer (InnoSpire Go, Phillips, Amsterdam, The Netherlands).

[0262] 2.3.3 Stability to freeze-thaw cycles. 100 µl of 0.25 mg / mL ISVD solution was subjected to 5 or 10 freeze-thaw (F / T) cycles. The solution was frozen at -20°C and thawed at room temperature.

[0263] 2.3.4 Concentration Measurement 。 Concentration and UV spectra were measured using a spectrophotometer (NanoDrop 2000 / 2000c, ThermoFisher). Concentration was determined based on absorbance at 280 nm. The mass extinction coefficients of NbE201 and NbE201-Cter used in the measurements were 1.888 and 1.716 (mg / mL), respectively. -1 cm -1 For ISVDs exposed to physical stress, concentrations were read before and after centrifugation at 10000g for 10 minutes at 4°C.

[0264] 2.3.5 Aggregate detection. Protein aggregates were visualized using nanoparticle tracking analysis (NTA) (ZetaView, 220 Twin Laser, Particle Metrix, Ammersee, Germany), a technique used to measure the size and concentration of individual particles. NbE201, NbE201-PEG10, and NbE201-PEG40 solutions (0.25 mg / ml) were analyzed before and after nebulization. Eleven locations were analyzed using the following parameters: Laser wavelength: 488 nm Scattering, no fluorescence Sensitivity: 80 Shutter speed: 100

[0265] 2.4 Preparation of dry powder formulations by spray drying method

[0266] Inhalation dry powders were formulated using NbE201 and NbE201-PEG40. The spray dryer used was a PROCEPT modular 4M8-Trix. The powder composition was: 65% trehalose dihydrate, 15% trileucine, 9.8% phosphate buffer, 0.2% rhodamine b, and 10% NbE20; or 37% trehalose dihydrate, 15% trileucine, 9.8% phosphate buffer, 0.2% rhodamine b, and 38% NbE201-PEG40 (10% NbE201 fraction and 28% PEG fraction). The spray drying conditions used were as follows: Flow rate: 10 ml / min Compressed air: 1.1 bar Inlet temperature: 145°C Outlet temperature: 51.1°C Relative humidity: 32% Nozzle size: 1.2 mm

[0267] 2.5 Pharmacokinetic studies.

[0268] 2.5.1. Mouse infusion and BAL collection. One nanomolar of NbE201, NbE201-PEG40, or AAT (Bio-Techne) was instilled intratracheally into wild-type Swiss mice and β-ENaC-overexpressing Tg mice in a final volume of 25 µl. Bronchoalveolar lavage fluid (BAL), nasal lavage fluid (NAL), blood samples, and lungs were collected at 0, 4, or 24 hours post-instillation. BAL and NAL samples were collected with 2 ml and 1 ml of Hanks' Balanced Salt Solution (HBSS) buffer, respectively. Cells from BAL and NAL samples were removed by centrifugation at 300 g for 5 min, and the supernatant was stored at -20°C. Lungs were collected in 0.5 ml of HBSS buffer, homogenized (Precellys evolution, Bertin Technologies, Montigny-le-Bretonneux, France), and centrifuged at 300 g for 15 min. The supernatant was stored at -20°C. Blood was collected via cardiac puncture and stored in tubes containing anticoagulant (Microvette® 500, K3 EDTA, VWR) at -20°C. All samples were treated with an antiprotease mixture (Roche).

[0269] The β-ENaC mouse model mimics the pathology of cystic fibrosis (CFibrosis) lungs and is currently the most representative mouse model because knockout mice of the CFibrosis Transmembrane Conductivity Regulator (cftr) gene (- / -) do not develop CFibrosis-related lung disease (Clarke et al., 1992, Sciences, 257(5073): 1125-8. Doi: 10.1126 / science.257.5073.1125). This will allow for the assessment of the effects of local chronic inflammation and the presence of thick mucus in the airway on the pharmacokinetics of the compounds under investigation.

[0270] 2.5.2. ELISA analysis. ISVD and AAT levels in BAL collected in vivo were measured using a sandwich ELISA method. ISVD analysis was performed using a Meso Scale Discovery (Rockville, USA) ELISA conversion kit, following the manufacturer's instructions. Uncoated Mesoscale compatible plates were coated with an anti-histidine-tagged antibody (BioLegend, San Diego, USA). ISVD was then detected using a mixture of anti-VHH antibody (Jackson ImmunoResearch, Ely, UK) and anti-goat Sulfo-TAG-tagged antibody (Meso Scale Discovery), and the signal was read using a MESO QuickPlex SQ 120MM (Meso Scale Discovery). AAT levels in the samples were measured using a commercial ELISA kit (Human Serpin A1 DuoSet ELISA, R&Dsystem, Mineapolis, USA) following the manufacturer's instructions.

[0271] 3. Protective activity of NbE201 against hNE-induced acute lung injury syndrome in mice.

[0272] 3.1. Animal Experiment Protocol

[0273] The in vivo efficacy of NbE201 was determined using a mouse model of acute lung injury induced by intranasal HNE instillation. To achieve this, female C3H x C57BL / 6 mice (wild-type littermates of a cystic fibrosis (CF)-like mouse model, overexpressing the β subunit of ENaC protein) were anesthetized by intraperitoneal injection of xylazine / ketamine (10 / 100 mg / kg, 0.1 ml / 10 g), followed by intranasal instillation of 15 µL of hNE solution (Elastin Products Company, USA; 25 µg / mouse = 0.8 nmol / mouse) or 15 µL of physiological saline (0.9% NaCl). Fifteen minutes prior to hNE treatment, nasal instillation of (30 µL) of Nb (Nb / HNE molar ratio of 4 / 1 nmol) or 0.9% NaCl was administered. Four hours after hNE infusion, mice were euthanized by intraperitoneal injection of pentobarbital solution (200 mg / kg, 0.1 mL / 10g).

[0274] 3.2. Bronchoalveolar lavage (BAL)

[0275] The trachea was exposed and clamped. The left lung was also clamped to allow for simultaneous pathological analysis of the un-lavaged lung. The lung was flushed three times with 1 mL of sterile saline (0.9% NaCl), and the BAL stream was collected. After centrifugation (1500 rpm, 4°C, 10 min), the total cells were resuspended in 200 µL of saline and counted in a Neubauer chamber using Turk solution. Cell smears were prepared from the BAL cells (900 rpm, 5 min) and stained with the Diff-Quick kit. The percentages of macrophages, eosinophils, lymphocytes, and neutrophils in the BAL stream were obtained by counting 200 cells based on their morphological characteristics. The supernatant of the BAL stream was aliquoted and frozen at -80°C for further analysis.

[0276] 3.3. Morphological Analysis

[0277] Unlaved lung tissue was fixed with 4% paraformaldehyde and embedded in paraffin. Sections were cut to 5 µm thickness using a microtome, and the dewaxed sections were stained with hematoxylin and eosin using standard histological techniques. Pathological studies were conducted to characterize the inflammatory response and the degree of tissue damage. The intensity of cell infiltration and tissue damage was scored to allow for intergroup comparisons and to quantify the beneficial protective effect of NbE201 treatment.

[0278] 3.4. Pro-inflammatory cytokines and chemokines

[0279] Concentrations of CC motif chemokine ligand 2 (CCL2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and keratinocyte-derived chemokines (KC) in BAL streams were measured using an ELISA kit (R&D Systems). Analysis was performed according to the manufacturer's instructions. Intergroup comparisons allowed for assessment of the anti-inflammatory protection provided by NbE201 pretreatment in animals.

[0280] 3.5 BAL hemoglobin concentration

[0281] The hemoglobin assay was performed using a Sigma kit according to the manufacturer's instructions. This kit is based on the Triton / NaOH method, which involves converting hemoglobin into heme complexed with Triton. Quantification of this stable complex was allowed by measuring the optical density at 400 nm. Intergroup comparisons allowed for assessment of the magnitude of hNE-induced intrabronchial and alveolar hemoglobin release and the protective effect of NbE201 pretreatment in mice.

[0282] 3.6 Quantitative Analysis of Alveolar-Capillary Injury

[0283] Lung tissue sections were digitized using panoramic scanning 2 (3DHISTECH). Image analysis was then performed using Visiopharm® software (v2022.01.3). This semi-automated analysis quantifies the damaged alveolar surface. These lesions are characterized by the presence of red blood cells in the alveolar interstitial spaces. Several steps were employed to achieve this ( Figure 3 A). The percentage of damaged surface area is determined by the following ratio: % Damaged area = (Damaged area / Alveolar area) × 100

[0284] 3.7 Statistical Analysis

[0285] Data are expressed as mean ± standard deviation (SD) (GraphPad Prism, v9). Statistical comparisons were performed using the Kruskal-Wallis test and Dunn's post-hoc test. A p-value less than 0.05 was considered significant.

[0286] Example 1 – Selecting NbE201

[0287] The immune library constructed from alpaca blood immunized with hNE was 4.6 x 10⁻⁶ in size. 7 The percentage of clones containing plasmids carrying the Nb gene was 75%. Genes from 91 positive clones from CE-ELISA were sequenced. Based on their CDR3 sequences, ISVDs with unique sequences were divided into 9 families, as shown in Table 1.

[0288]

[0289] Table 1: ISVD families selected in solution by directional sandwich panning, and the names of ISVDs belonging to these families. Within a family, all ISVDs share the same CDR3, but their sequences differ in the region outside that CDR. This means that these ISVDs are expected to bind to the same epitopes, despite differences in affinity.

[0290] One ISVD from each family was expressed and purified in *E. coli* WK6. The ability of pure ISVDs to inhibit hNE activity was evaluated at an ISVD:hNE ratio of 4:1. Only NbE201 inhibited hNE (residual activity 3 ± 0.03%). The NbE201 gene was transformed into the expression vector pHEN6 for mass production in *E. coli* WK6 cells.

[0291] The amino acid sequence encoded by NbE201 is as follows:

[0292] The sequences of the three CDRs are as follows:

[0293] Example 2 - Production and Purification of NbE201

[0294] Following expression in the periplasm of E. coli WK6, several independent batches of NbE201 were purified to homogeneity. SDS-PAGE analysis confirmed that the purity of each batch was above 95%, and mass spectrometry confirmed the integrity of the protein (data not shown).

[0295] Example 3 – NbE201 is a tight inhibitor of hNE

[0296] 3.1 Inhibiting the hydrolysis of small molecule synthesis substrates The substrate (N-succinyl-Ala-Ala-Ala-p-nitroaniline) Km was calculated by incubating hNE with different concentrations of the substrate. The initial rate v0 of hNE hydrolysis was plotted against [S] using the Hanes-Woolf linearization method: [S] / V0 vs [S]. The obtained Km... m It equals 1.4 mM.

[0297] The substrate (MeOSuc-Ala-Ala-Pro-Val-AMC) Km was calculated by incubating mNE with different concentrations of substrate. The initial rate v0 of mNE hydrolysis was plotted against [S] using the Hanes-Woolf linearization method: [S] / V0 vs [S]. The obtained Km... m It equals 0.478 ± 0.044 mM.

[0298] The residual activity of hNE in the presence of different concentrations of NbE2O1 was determined by the initial rate of substrate hydrolysis. Figure 4 When the concentration of NbE201 present was similar to (i.e., within 10-fold) the total enzyme concentration (50 nM), the activity of hNE was significantly inhibited. This observation indicates that NbE201 acts as a tightly bound inhibitor. The IC50 for inhibiting the hydrolysis of N-succinyl-Ala-Ala-Ala-p-nitroaniline by hNE was calculated. 50 The concentration was 38.3 ± 3.3 nM (hNE concentration: 50 nM). K i It is through the three K values ​​of three independent curves. i The values ​​are calculated by averaging, and each curve represents an experiment conducted using different batches of production Nb. Based on each curve, v0 / v is plotted. i The graph of [NbE201] is compared, and K is estimated using the following formula. i : The Morrison formula is established for tight-bonded agents and is defined as follows:

[0299] Where V i V0 and V0 represent the initial rates measured in the presence or absence of inhibitors, respectively; [E] T This represents the total enzyme concentration; [I] T This represents the total inhibitor concentration; To correct the substrate concentration relative to K m Before the inhibition mode-specific effect, the apparent inhibition constant K i The apparent dissociation constant of the enzyme-inhibitor complex. The Ki of mNE was obtained using the same strategy. Table 2 lists the Ki values ​​for both enzymes.

[0300] This confirms that NbE201 is a tight-binding inhibitor. Similar inhibitory effects were observed when NbE201 was pre-incubated with hNE for 15 minutes before adding the substrate. Furthermore, the experiment was performed in microplate form (100-second time limit) or in 1.5 mL wells (14-second time limit). In both cases, the graphs did not show bending at the initial time point, indicating that the equilibrium between the enzyme, substrate, and NbE201 is reached within seconds.

[0301] For comparison, the inhibitory effects of three other inhibitors on the hydrolysis of N-succinyl-Ala-Ala-Ala-p-nitroaniline (hNE) were analyzed: AAT (α-1-antitrypsin, Sigma-Aldrich, Germany, covalent natural inhibitor), elastase inhibitor 3 (EI3, Calbiochem, Merck Millipore, Darmstadt, Germany, small molecule, covalent inhibitor), and cevelexstat (R&D systems, UK, small molecule, non-covalent inhibitor). The measured IC50 values ​​were 28 nM, 349 ± 70 nM, and 77 ± 1 nM, respectively. The IC50 of NbE201 was in the same range as AAT and superior to that of cevelexstat, the only clinically used small, non-competitive inhibitor.

[0302] 3.2. Inhibit the hydrolysis of the natural substrate elastin. The activity of hNE in inhibiting the hydrolysis of the physiological substrate elastin was measured using NbE201 and other hNE inhibitors (i.e., α-1-antitrypsin (AAT, Sigma-Aldrich, Germany), elastase inhibitor 3 (EI3, Calbiochem, Merck Millipore, Darmstadt, Germany), and cevelextastat (R&D systems, UK)). To determine the residual activity of hNE, the absorbance of samples with different inhibitor:hNE ratios was compared with the absorbance measured without inhibitors, such as... Figure 5 As shown, when the [NbE201]:[hNE] ratio was 15, hNE activity was inhibited by 80%. AAT showed higher inhibitory activity against hNE than NbE201 (85% inhibition at an [AAT]:[hNE] ratio of 1.45). When the [EI3]:[hNE] ratio was 15, hNE activity was inhibited by 95%. These differences can be explained by the inhibitory mechanisms of the tested inhibitors. AAT and EI3 are covalent inhibitors, while NbE201 inhibits hNE non-covalently. Cevillasestat is a non-covalent hNE inhibitor; when [Cevillasestat]:[hNE] = 50, the inhibition rate was only 40%, while NbE201 showed almost zero residual hNE activity at the same ratio. The IC50 values ​​for NbE201, AAT, EI3, and cevelexat were 487.75 ± 70 nM, 120 ± 4 nM, 271 ± 28 nM, and 7760 ± 255 nM, respectively. Therefore, NbE201 exhibits stronger inhibitory activity against the hydrolysis of physiological substrates than cevelexat, the only clinically used small molecule inhibitor.

[0303] Example 4 - Determination of the affinity of NbE201 for hNE.

[0304] Quantitative measurements were performed to measure the kinetic constant (k). on , k off ) and equilibrium constant (K D The obtained sensor image is as follows: Figure 6 As shown in the table. A 1:1 ligand model was used to globally fit the kinetics obtained from five different concentrations of hNE. Table 2 lists the kinetic constants and equilibrium constants characterizing the binding.

[0305] Example 5 - NbE201 is NE-specific.

[0306] The ability of NbE201 to inhibit other serine proteases was verified using chromogenic substrates. Figure 7 A). This data indicates that NbE201 does not inhibit human cathepsin G (CG, with 36.7% identity to hNE) and porcine pancreatic elastase (pPE, with 39.2% identity to hNE), while NbE201 can inhibit human protease 3 (PR3, with 54.7% identity to hNE), but the inhibition efficiency is very low (20% inhibition at an NbE201:enzyme ratio of 60). More importantly, Figure 7 B showed that NbE201 significantly inhibited mouse elastase (mNE, with 74.9% sequence identity to hNE). IC 50 It is 49.4 ± 6.6 nM.

[0307] To confirm that NbE201 is hNE-specific and does not bind to other serine proteases, BLI assays were performed in the presence of five different proteases (hNE, hCG, hPR3, mouse pancreatic elastase (mPE), and porcine pancreatic elastase (pPE)). Figure 8 The results showed that a binding signal only appeared when NbE201 came into contact with hNE, meaning that NbE201 does not bind to other proteases. This confirms its high specificity for NE.

[0308] Furthermore, the affinity between NbE2O1 and mNE was measured using BLI, such as... Figure 9 As shown. For hNE, a 1:1 ligand model was used to fit the sensor plot. The kinetic and equilibrium binding parameters are listed in Table 2.

[0309]

[0310] Table 2: K D k on and k offThe values ​​describe the binding between NbE201 and either hNE or mNE. Experiments were independently replicated (N=2 for hNE, N=1 for mNE), and the mean values ​​(±SD) of the measured kinetic parameters are shown. The K values ​​for hNE (N=3) and mNE (N=6) as determined by enzymatic assay are also shown. i .

[0311] This indicates that the binding affinity of NbE201 to hNE is about 10 times higher than that to mNE, and this includes an inhibitory activity against hNE that is about 10 times higher than that against mNE.

[0312] Example 6 - NbE201 is a competitive inhibitor of NE.

[0313] To determine whether NbE201 binds to or near the active site of elastase, competitive binding experiments were performed using molecules known to bind to the active site of elastase. The first competitive molecule tested was AAT, a natural inhibitor of hNE. Figure 10 (Right figure) shows that hNE, which forms a complex with AAT, does not bind to NbE201, indicating that NbE201 binds to the active site of hNE. AAT forms a covalent complex with hNE, and both AAT and hNE undergo conformational changes after complex formation. Therefore, the hNE-AAT pre-formed complex cannot bind to NbE201, possibly because this conformational rearrangement results in the NbE201 epitope no longer being present on the hNE surface. To confirm this, two other known inhibitory molecules that can bind to the active site of hNE were tested. These molecules are: (i) MeOSuc-AAPV-CMK, also known as elastase inhibitor 3 (EI3) and methyl 4-[[(2S)-1-[[(2S)-1-[(2S)-2-[[(3S)-1-chloro-4-methyl-2-oxopentane-3-yl]carbamoyl]pyrrolidine-1-yl]-1-oxopropane-2-yl]amino]-1-oxopropane-2-yl]amino]-4-oxobutyrate, a small molecule inhibitor that covalently binds to the hNE active site but does not induce a conformational change in hNE; and (ii) cAb-H7S-Nter-P1, which is an ISVD without hNE binding fused to an inhibitory peptide (P1) that non-covalently binds to the hNE active site. Figure 10The results showed that when hNE was pre-incubated with saturated concentrations of both inhibitors, it failed to bind to NbE201. While the competition between NbE201 and AAT or Nb-H7S-Nter-P1 may be due to steric hindrance (i.e., NbE201 binds near the active site, thus exhibiting binding to the active site-targeting molecule), the strong competition with the small hNE competitive inhibitor (MeOSuc-AAPV-CMK) strongly indicates that NbE201 binds to the active site of hNE.

[0314] Example 7 - Stability of NbE201 to urea-induced development.

[0315] Through intrinsic fluorescence (λ) max and F 337nm The thermodynamic stability of NbE201 was investigated by monitoring urea-induced expansion using both 3D and far-UV-CD (207 nm) methods. A single transformation was obtained in both cases, indicating that both the tertiary and secondary structures follow a two-state model (N...). U) Collaborative Deployment ( Figure 11 The reversibility of denaturation after urea concentration dilution was established using intrinsic fluorescence. The coincidence of the conversion curves obtained from intrinsic fluorescence and far-UV-CD measurements after normalization indicates that secondary and tertiary structures unfold simultaneously. Figure 12 Therefore, protein denaturation can be modeled using the epigenetic cooperative two-state model (N). The model is described by U), where only the natural and expanded states are significantly occupied. Therefore, assuming a simple two-state model and Equation 1, the values ​​of the thermodynamic parameters are calculated by monitoring the transitions using three parameters, as shown in Table 3. ΔG ° NU(H2O) The value is in the higher range of other observed Nb values ​​(Dumoulin et al., 2002, doi: 0.1110 / ps.34602).

[0316]

[0317] Table 3: The thermodynamic parameters of NbE201 at pH 7 and 25°C were obtained by analyzing the urea-induced equilibrium transition.

[0318] Example 8 - Stability of NbE201 against thermally induced denaturation.

[0319] The stability of NbE201 to thermally induced unfolding was assessed by heating protein samples from 25°C to 97°C and then cooling them back to 25°C, followed by measuring the signal at 206 nm using far-UV-CD. Figure 13 This experiment used three NbE201 samples from three independent batches. Figure 14A single transformation was obtained, indicating that the secondary structure follows a two-state model (N). U) Co-unfolding. Although the degree of folding is very significant after the temperature decreases, folding only occurs partially; therefore, only the apparent unfolding temperature (T) is considered. m app (i.e., the temperature at which 50% of the molecules unfold while the rest remain in their native state). The Tm was calculated and averaged for the conversions obtained from three different batches of ISVD, which was 70.7 ± 1.5°C, a value that is in the high range of observed Tm values ​​for ISVD (Dumoulin et al., 2002, doi: 10.1110 / ps.34602).

[0320] Example 9 - Stability of NbE201 to human neutrophil elastase.

[0321] The stability of NbE201 against hNE protein hydrolysis activity was tested after 14 days. The stability of the complex was assessed by SDS-PAGE. Figure 15 The percentage of the remaining complex over time is shown (upper curve). The complex remains at approximately 100% over time, meaning that NbE201 is stable and not digested within two weeks in the presence of hNE.

[0322] To ensure that the stability of ISVD was not due to the complete loss of enzyme activity, hNE activity was also measured within 14 days. Figure 15 The curves showing the percentage of remaining activity of hNE are shown (lower curve). Over time, hNE loses some of its activity. However, this loss of activity cannot explain approximately 100% of the complex stability. In summary, these data indicate that NbE201 is stable for 14 days in the presence of hNE.

[0323] Example 10 - Crystal structure of NbE201 and the complex NbE201-hNE.

[0324] The NbE201-hNE complex was crystallized in group P212121 with a final resolution of 2.3 Å. The asymmetric unit contained a single NbE201-hNE complex. The structure showed a clear density of amino acids I1 to Q218 of hNE, except for amino acids R147 and N148 according to the prochymotrypsinogen numbering scheme; amino acids Q1 to S123 of NbE201 also showed a clear density according to the IMGT numbering scheme. Table 4 shows the refined statistics. According to the prochymotrypsinogen numbering scheme, we found glycans in the NAG-NAG-FUC conformation at sites N109 and N204.

[0325]

[0326] Table 4: Data collection and detailed statistics of X-ray structures of NbE201 complex with hNE and NbE201 alone.

[0327] The interaction between CDR3 of NbE201 and hNE is mainly mediated by polar interactions between amino acids P100, K101, D107, Y110, and Y112 of NbE201 (located at the boundary between CDR3 and the framework) and amino acids S195, A60, R36, N61, and P96 of hNE. Figure 16 A). At the CDR1 level, there is only one interaction between S30 of NbE201 and G218 and G219 of hNE. Furthermore, hydrophobic interactions are limited to V2 of NbE201 and V97 of hNE. Figure 16 B). Proline P100, belonging to CDR3, establishes a polar interaction with the hNE active site S195 via water molecules. Figure 17 A). On the other hand, the amino acids H57 and D102 of the enzyme's catalytic triplet do not bind to NbE201 ( Figure 17 A). Therefore, the inhibitory effect of Nb E201 on hNE activity is likely due to its interaction with serine S195 through water molecule interactions. The structural resolution of the ISVD alone is 1.8 Å ( Figure 16 C). Superimposing the individual NbE201 structure with the structure of the Nb-hNE complex revealed no structural differences.

[0328] Example 11 - PEGylation of NbE201 does not affect its inhibitory ability.

[0329] Following the previously described protocol, NbE201 was PEGylated by attaching polyethylene glycol (PEG) to a maleimide functional group, which is capable of reacting with a C-terminal cysteine ​​residue. Three types of PEG were used: linear PEG at 10 or 20 kDa, or branched PEG at 40 kDa. The PEGylated products were purified by FPLC on a cation exchange column, and their purity was verified by electrophoretic gel staining. The inhibitory effect on purified hNE was then evaluated by spectrophotometric measurement of the cleavage of the hNE substrate in the presence or absence of Nb. Figure 18 Inhibition profiles of wild-type NbE201 and its PEGylated forms were obtained by exposing hNE to ISVD in an in vitro enzymatic assay, with a molar ratio of hNE:NbE201 ranging from 1:0.25 to 1:5. No significant difference was observed in the inhibitory activity between NbE201 and its PEGylated counterparts.

[0330] Example 12 - PEGylation protects Nb from damage caused by atomization, stirring and freeze-thaw cycles

[0331] To assess whether PEGylation increases or decreases the stability of NbE201, the resistance of natural and PEGylated ISVDs to different stresses was investigated and compared. Therefore, wild-type and PEGylated ISVDs were exposed to atomization, magnetic stirring, F / T cycling, and exposure to chemical denaturants and heat. So far, wild-type NbE201 and PEG10-modified NbE201 (PEG10 or 40) were exposed to MeSH atomization, 1 or 4 hours of stirring, and 5 or 10 F / T cycles. To highlight the presence of insoluble aggregates, their concentrations before and after centrifugation at 10,000 g were measured. For non-PEGylated ISVDs, a concentration decrease was observed in stirred and atomized samples before centrifugation (Table 5). This decrease was more pronounced in centrifuged samples. A concentration decrease after centrifugation was also observed in samples exposed to F / T cycling. However, this concentration loss was not observed in NbE201-PEG40 samples exposed to stirring or F / T cycling (Table 5). Although a decrease in NbE201-PEG40 concentration was observed in the nebulized samples, no difference was detected after centrifugation. This suggests that the concentration decrease may not necessarily be due to protein aggregation, but rather to protein loss during the nebulization and sampling processes. Similar results were obtained using NbE201-PEG10, indicating the protective effect of PEGylation.

[0332]

[0333] Table 5. Percentage of preserved ISVD concentration measured by absorbance at A280 nm before and after centrifugation at 10000 g for 10 minutes. Initial percentage of wild-type and PEGylated (linear PEG10 or branched PEG40) NbE201 concentration after exposure to nebulization, magnetic stirring for 1 or 4 hours, or 5 or 10 freeze-thaw (F / T) cycles. N=3, n=3 (NbE201wt and NbE201-PEG40). N=2, n=3 (NbE201-PEG10). N=number of experiments, n=number of technical replicates. Mean ± SD. Statistical tests (NbE201wt and NbE201-PEG40): one-way ANOVA followed by Dunnett's post-hoc test, compared with the corresponding control group. P-value < 0.05 P-value < 0.01 P-value < 0.001 P-value < 0.0001.

[0334] Then, the inhibition capacity of samples exposed to nebulization, stirring, or F / T cycling was compared with that of unexposed ISVD (Table 5 and 1). Figure 19 The observed loss of inhibitory activity induced in wild-type NbE201 was consistent with the concentration loss described above, as the largest loss was observed after 4 hours of nebulization and agitation. However, the smaller concentration loss described above was not correlated with the decrease in inhibitory activity in samples exposed to F / T cycles. The NbE201-PEG40 sample did not show any significant loss of inhibitory activity, indicating that ISVD has increased stability to applied stress due to PEGylation. Similar results were obtained using NbE201-PEG10. To our knowledge, there is currently no description of PEGylation for preventing nebulization.

[0335] The presence of aggregates was confirmed by NTA analysis of the nebulized samples and their controls. Particle size and number of NbE201, NbE201-PEG10, and NbE201-PEG40 were measured before and after nebulization. To ensure measurement reliability, at least 50 particles were counted per frame on average. Except for nebulized NbE201, the average particle count in the blank solution and all measurement solutions was well below the threshold, while the average particle count in nebulized NbE201 was 228 per frame. The 10th, 50th, and 90th percentiles of the counted particles were 22.3 ± 12, 138 ± 8, and 342.8 ± 26 nm, respectively. Therefore, this method can track particles in nebulized natural NbE201 samples but not in nebulized PEGylated NbE201 samples.

[0336] Example 13 - Dry powder formulation.

[0337] Since NbE201 is intended to treat lung diseases, an inhalation dry powder was prepared using NbE201 and NbE201-PEG40 and characterized. Two protein stabilizers were added to the powder composition: trehalose (a non-reducing sugar) and trileucine.

[0338] The powder was dissolved in water and then dialyzed into phosphate buffer (50 mM, pH 7) to assess the integrity of ISVD in the spray-dried powder. To detect the presence of insoluble aggregates, protein concentration was measured by reading absorbance at 280 nm before and after centrifugation (10000 g, 10 min). For NbE201, the concentration read after centrifugation was 53 ± 3% of the concentration read before centrifugation, indicating that almost half of the Nb aggregated during spray drying. Conversely, the concentration of NbE201-PEG40 after centrifugation was 97 ± 2% of the initial concentration, indicating that PEGylation prevented aggregation during spray drying.

[0339] The inhibitory activity was further evaluated after the powder was dissolved. In an in vitro assay, the inhibitory capacity of Nb was assessed at a [hNE]:[ISVD] ratio of 1:1. The inhibitory capacity of the dissolved powder was compared with that of its corresponding controls (e.g., untreated NbE201 or NbE201-PEG40). While NbE201 showed a decrease in inhibitory capacity at the studied ratio due to spray drying, this was not the case with NbE201-PEG40 (Table 6). Furthermore, the inhibitory capacity of the spray-dried ISVD was evaluated after centrifugation to determine whether the remaining Nb (without aggregates) exhibited a decrease in inhibitory capacity. The protein concentrations used to calculate the hNE:ISVD ratio were those read after centrifugation. Under these conditions, no decrease in inhibitory activity was observed in either ISVD, indicating that the soluble ISVD retained its inhibitory capacity.

[0340]

[0341] Table 6: Percentage of hNE inhibition in untreated (control) NbE201 or NbE201-PEG40 or spray-dried NbE201 or NbE201-PEG40 before or after centrifugation at 10000g to remove aggregates. N=1, n=3.

[0342] Example 14 - NbE201 can inhibit the proteolytic activity in the sputum of patients with cystic fibrosis, and PEGylation prolongs its activity.

[0343] The inhibitory effect of NbE201 on sputum from patients with cystic fibrosis was then investigated. First, inhibition curves for individual ISVDs were obtained by exposing 20 mg of sputum to amounts ranging from 0 to 60 µg of NbE201. The amount of NbE201 required to completely inhibit sputum proteolytic activity varied depending on the visual classification of the sputum (mucus, pus, or mucopurulent) and previous proteolytic activity estimated based on standard curves of different amounts of purified hNE. The amount required to inhibit proteolytic activity in purulent samples was higher than that required to inhibit proteolytic activity in mucopurulent samples. Figure 20 ).

[0344] The inhibitory activity of NbE201 in sputum was then compared with that of PEGylated ISVD and AAT. 15 mg of sputum was exposed to concentrations of 0–15 µmol / ml of NbE201, NbE201-PEG10, NbE201-PEG40, or AAT, and inhibition curves were obtained. Figure 21Although a 1:1 AAT:hNE ratio is sufficient to completely inhibit elastase in aqueous solutions, the inhibitory effect of AAT in sputum is lower than that of PEGylated and native ISVD.

[0345] Nonlinear regression curves were fitted using GraphPad Prism 9.1.2 to interpolate Nb / AAT concentrations, thereby inhibiting 50% of sputum activity. Elastase activity per sputum sample was then estimated based on a standard curve constructed using incremental concentrations of purified hNE. The molar ratio of nanobody / AAT:hNE inhibiting 50% of sputum proteolytic activity was then calculated based on the estimated hNE activity in the sputum. Figure 22 Although AAT only requires a 1:1 AAT:hNE molar ratio to achieve complete elastase inhibition in vitro, while a 3:1 NbE201:hNE ratio is needed, the AAT:hNE ratio required to inhibit 50% of a given sputum volume is significantly higher than that of NbE201:hNE. No significant difference was observed between native ISVD and PEGylated ISVD.

[0346] The inhibitory capacity of unPEGylated and PEGylated Nb was investigated over time. To this end, the inhibitory capacity was assessed after different exposure times from 0 to 24 hours. At time 0, no significant difference was observed between the inhibitory effect induced by NbE201 and that induced by NbE201-PEG10 or NbE201-PEG40. However, over time, a decrease in the inhibitory capacity of NbE201 was observed, while the loss of PEGylated ISVD was reduced. Figure 23 ).

[0347] The results of this study show that NbE201 can inhibit proteolytic activity in isolated sputum samples, and PEGylation can prolong the inhibitory effect.

[0348] The same experiment was then repeated to compare the inhibitory capacity of ISVD with that of AAT over time. Although the inhibitory capacity of AAT was lower at time 0, it did not show any significant decrease over time. Figure 23 ).

[0349] Example 15 – UnPEGylated and PEGylated NbE201 can inhibit ciliary dyskinesia and proteolytic activity in the sputum of COPD patients.

[0350] Then, the inhibitory effects of NbE201(-PEG40) or AAT on sputum in patients with COPD exacerbations and sputum in patients with ciliary dyskinesia (CD) were investigated.

[0351] Inhibition profiles were obtained by exposing 15 mg (COPD) or 10 mg (CD) sputum to increasing concentrations of NbE201, NbE201-PEG40, or AAT. No significant difference in inhibitory activity was observed between PEGylated and natural ISVD and AAT. Figure 25 and 26 ).

[0352] Example 16 - PEGylation increases the residence time of NbE201 in β-ENaC and wild-type Swiss mice.

[0353] Pharmacokinetic studies were conducted in wild-type Swiss mice and β-ENaC mouse models simulating the pathophysiology of cystic fibrosis. NbE201, NbE201-PEG40, and AAT were instilled intratracheally, and bronchoalveolar lavage (BAL) was collected at 0, 4, or 24 hours. ISVD and AAT were then quantified by ELISA. PEGylation with PEG40 was observed to increase the residence time of NbE201. In β-ENaC and Swiss mice, only 3.3% and 4.4% of the NbE201 dose collected at time 0 were quantified at 24 hours, respectively; while in β-ENaC and Swiss mice, 56% and 65% of the NbE201-PEG40 dose collected at time 0 were quantified at 24 hours, respectively. AAT quantification in BAL collected at 24 hours was moderate, with 25% and 33% of the dose collected at time 0 recovered in β-ENaC and Swiss mice, respectively. No significant differences were observed between the two mouse models. Figure 27 ).

[0354] Example 17 – Protective effect of NbE201 against acute lung injury syndrome in an hNE-induced mouse model.

[0355] The presence of hemoglobin reflects the occurrence of alveolar capillary lesions following hNE infusion. Figure 3 B and 3C). Histologically, "hemorrhagic lesions" defined by erythrocyte-rich areas were observed in the unwashed lungs. Figure 3 D). We have developed and characterized a relevant mouse model (nasal infusion of hNE 25 µg), which allows us to evaluate the effects of the selected NbE201 by analyzing alveolar capillary damage. Like AAT, NbE201 protects the lungs from alveolar capillary damage in the presence of hNE. Figure 3 D).

Claims

1. A binding agent capable of specifically binding to and inhibiting neutrophil elastase (NE), wherein The binder comprises an immunoglobulin single variable domain (ISVD), and the binder competes with elastase inhibitor 3 (EI3) for binding to NE.

2. The binding agent according to claim 1, wherein the binding agent comprises an immunoglobulin single variable domain (ISVD) that binds to at least one of the R36, A60, N61, P96, V97, S195, G218, and G219 residues of NE, preferably binding to two or more of the R36, A60, N61, P96, V97, S195, G218, and G219 residues of NE, more preferably binding to three or more of the R36, A60, N61, P96, V97, S195, G218, and G219 residues of NE, and even more preferably binding to all of the R36, A60, N61, P96, V97, S195, G218, and G219 residues of NE, for example, binding to at least the R36, A60, N61, P96, V97, and S195 residues of NE.

3. The binding agent according to claim 1 or 2, wherein the binding agent comprises an immunoglobulin single variable domain (ISVD), the ISVD comprising a complementarity-determining region 1 (CDR1) having the sequence shown in SEQ ID NO: 1 (GRTISLYR), a CDR2 having the sequence shown in SEQ ID NO: 2 (INWSGDMT), and a CDR3 having the sequence shown in SEQ ID NO: 3 (TADPKLLPLADSSYGY).

4. The binder according to claim 1 or 2, wherein the binder comprises an ISVD, the ISVD comprising CDR1, CDR2 and CDR3, each CDR being present in SEQ ID NO: 4, wherein CDR1, CDR2 and CDR3 are annotated according to any one of the numbering systems IMGT, Kabat, Chlotia, Martin or AHo.

5. The binder according to claims 1 to 4, comprising or consisting of VHH.

6. The binder according to any one of claims 1 to 5, wherein the ISVD comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 (QVQLQESGGGLVQAGGSLRLSCVVPGRTISLYRMGWFRQAPGKEREFVAGINWSGDMTDYVDSVKGRFTISRDNAKNTVYLEMNSLKPEDTAIYYCTADPKLLPLADSSYGYWGQGTQVTVSS).

7. The binder according to any one of claims 1 to 6, wherein the ISVD comprises the amino acid sequence shown in SEQ ID NO: 4(QVQLQESGGGLVQAGGSLRLSCVVPGRTISLYRMGWFRQAPGKEREFVAGINWSGDMTDYVDSVKGRFTISRDNAKNTVYLEMNSLKPEDTAIYYCTADPKLLPLADSSYGYWGQGTQVTVSS).

8. The binder according to any one of claims 1 to 7, wherein the binder binds to at least a portion of the active site of the NE.

9. A nucleic acid molecule comprising a polynucleotide sequence encoding a binding agent according to any one of claims 1 to 8, or a vector comprising the nucleic acid molecule.

10. A cell or virus comprising a nucleic acid molecule or vector according to claim 9, optionally wherein the cell is capable of expressing a binding agent or expressing a binding agent, or the virus is configured to induce expression of the binding agent in recipient cells infected by the virus.

11. A pharmaceutical composition comprising a binder according to any one of claims 1 to 8, a nucleic acid molecule or carrier according to claim 9, or a cell or virus according to claim 10, and a pharmaceutically acceptable carrier, diluent, and / or excipient, optionally wherein the pharmaceutical composition comprises other NE inhibitors.

12. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition is suitable for nasal or pulmonary administration to a subject, optionally wherein the pharmaceutical composition is formulated as an inhalable dry powder, optionally wherein the pharmaceutical composition is configured for use in a nebulizer or metered-dose inhaler.

13. An apparatus comprising the pharmaceutical composition according to claim 11 or 12, wherein the apparatus is a nebulizer, a metered-dose inhaler, or a dry powder inhaler.

14. A kit, such as a diagnostic kit, comprising a binder according to any one of claims 1 to 8, a nucleic acid molecule or vector according to claim 9, or a cell or virus according to claim 10.

15. The binder according to any one of claims 1 to 8, the nucleic acid molecule or carrier according to claim 9, the cell or virus according to claim 10, or the pharmaceutical composition according to claim 11, for pharmaceutical use.

16. Use of the binder according to any one of claims 1 to 8, the nucleic acid molecule or carrier according to claim 9, the cell or virus according to claim 10, or the pharmaceutical composition according to claim 11, for the prevention or treatment of inflammatory diseases.

17. Use of the binder, nucleic acid molecule, carrier, cell or virus, or pharmaceutical composition according to claim 16, wherein the inflammatory disease is a lung disease, preferably selected from diseases of mucoid obstructive pulmonary disease, such as cystic fibrosis, chronic obstructive pulmonary disease (COPD), bronchiectasis, ciliary dyskinesia and acute respiratory distress syndrome, inflammatory nasal and sinus diseases, or the inflammatory disease is inflammatory bowel disease, or the inflammatory disease is an inflammatory skin disease.

18. Use of the binder according to any one of claims 1 to 8, the nucleic acid molecule or carrier according to claim 9, the cell or virus according to claim 10, or the kit according to claim 14, for the diagnosis of inflammatory diseases in subjects.

19. A method for determining the level of active neutrophil elastase in a sample, the method comprising contacting the sample with a binder according to any one of claims 1 to 8, and detecting at least the neutrophil elastase bound to the binder.

20. A method for diagnosing, prognosing, and / or monitoring inflammatory diseases in subjects, optionally any inflammatory disease as defined in claim 17, the method comprising: - The biological sample obtained from the subject is contacted with the binder according to any one of claims 1 to 8, and - The level of active neutrophil elastase in the sample is determined by detecting at least the neutrophil elastase that is bound to the binder.

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